Material and methods for treating or preventing her-3 associated diseases

ABSTRACT

Described herein are materials and methods for treating subjects having a HER-3 associated disease, by administering a first agent that binds to HER-3, in combination with a second agent that binds and/or inhibits another member of the HER family. The first and the second agent may be a biologic, such as an antigen-binding protein, or a small molecular tyrosine kinase inhibitor, for example.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to provisionalpatent Application No. 61/261,149, filed Nov. 13, 2009.

BACKGROUND

1. Technical Field

This document relates to materials and methods for treating subjectshaving a disease associated with Human Epidermal Growth FactorReceptor-3 (HER-3) by administering a first agent that binds to HER-3,in combination with a second agent that binds or inhibits another HumanEpidermal Growth Factor Receptor (HER) family member. The first and thesecond agent may be any kind of molecule that binds to HER-3 or binds toand/or inhibits another HER family member, respectively, including, butnot limited to a biological compound, such as an antigen bindingprotein, a small molecular tyrosine kinase inhibitor, an siRNA, or anatural substance.

2. Background

HER-3, also known as ErbB3, is a receptor protein tyrosine kinase thatbelongs to the epidermal growth factor receptor (EGF-R, also known asHER) family of receptor protein tyrosine kinases, which also includesHER-1 (also known as EGF-R or erbB), HER-2 (also known as erbB2), andHER-4 (also known as erbB4) (Plowman et al. (1990) Proc. Natl. Acad.Sci. US 87:4905-4909; Kraus et al. (1989) Proc. Natl. Acad. Sci. US86:9193-9197; and Kraus et al. (1993) Proc. Natl. Acad. Sci. US90:2900-2904). Like the prototypical epidermal growth factor receptor,the transmembrane receptor HER-3 consists of an extracellularligand-binding domain (ECD), a dimerization domain within the ECD, atransmembrane domain (TMD), an intracellular protein tyrosine kinasedomain (TKD), and a C-terminal phosphorylation domain.

The ligand for HER-3, known as heregulin (HRG), binds to theextracellular domain of HER-3 and activates receptor-mediated signalingby promoting dimerization with other human epidermal growth factorreceptor (HER) family members, subsequent transphosphorylation of theintracellular HER-3 domain, and activation of downstream signalingcascades. Dimer formation with multiple HER family members expand thesignaling potential of HER-3, and is a means for signal diversificationas well as signal amplification.

SUMMARY

This document relates to materials and methods for treating subjectshaving an HER-3 associated disease, by administering an agent that bindsto HER-3, in combination with a second agent that binds to and/orinhibits another member of the HER family. The first and the secondagent may be any kind of molecule that binds to HER-3 or binds to and/orinhibits another HER family member, respectively, including, but notlimited to a biological compound, such as an antigen binding protein, asmall molecular tyrosine kinase inhibitor, an siRNA, or a naturalsubstance.

In one aspect, this document features a method of treating or preventinga disease associated with HER-3 in a subject, comprising administeringto the subject a first agent and a second agent, wherein the first agentbinds to HER-3 and the second agent binds to and/or inhibits theactivity of another member of the HER family. The first agent can be asmall molecule compound or an antigen-binding protein that binds toHER-3. The first agent can be an antigen-binding protein that binds toHER-3 and comprises a heavy chain amino acid sequence that comprises aCDRH1 selected from the group consisting of SEQ ID NOs:236, 251, 252,and 256; a CDRH2 selected from the group consisting of SEQ ID NOs:258,278, 280, and 282; and a CDRH3 selected from the group consisting of SEQID NOs:283, 285, 309, 313, and 315; and a light chain amino acidsequence that comprises a CDRL1 selected from the group consisting ofSEQ ID NOs:320, 334, 337, and 340; a CDRL2 selected from the groupconsisting of SEQ ID NOs: 343, 356, 351, and 344; and a CDRL3 selectedfrom the group consisting of SEQ ID NOs:360, 381, 385, and 387. Thefirst agent can be an antigen-binding protein that binds to HER-3 andcomprises a heavy chain amino acid sequence that comprises at least oneof the CDR's selected from the group consisting of (a) CDRH1's as shownin SEQ ID NOs:236, 251, 252, and 256; (b) CDRH2's as shown in SEQ IDNOs:258, 278, 280, and 282; and (c) CDRH3's as shown in SEQ ID NOs:283,285, 309, 313, and 315. The first agent can be an antigen-bindingprotein that binds to HER-3 and comprises a light chain amino acidsequence that comprises at least one of the CDR's selected from thegroup consisting of: (d) CDRL1's as shown in SEQ ID NOs: 320, 334, 337,and 340; (e) CDRL2's as shown in SEQ ID NOs:343, 356, 351, and 344; and(f) CDRL3's as shown in SEQ ID NOs:360, 381, 385, and 387.

The first agent can be an antigen-binding protein that binds to HER-3and comprises a heavy chain amino acid sequence that comprises at leastone of the CDR's selected from the group consisting of (a) CDRH1's asshown in SEQ ID NOs: 236, 251, 252, and 256; (b) CDRH2's as shown in SEQID NOs:258, 278, 280, and 282; and (c) CDRH3's as shown in SEQ IDNOs:283, 285, 309, 313, and 315; and a light chain amino acid sequencethat comprises at least one of the CDR's selected from the groupconsisting of: (d) CDRL1's as shown in SEQ ID NOs:320, 334, 337, and340; (e) CDRL2's as shown in SEQ ID NOs:343, 356, 351, and 344; and (f)CDRL3's as shown in SEQ ID NOs:360, 381, 385, and 387. The first agentcan be an antigen-binding protein that binds to HER-3 and comprises aheavy chain amino acid sequence that comprises a CDRH1 selected from thegroup consisting of SEQ ID NOs: 236, 251, 252, and 256, a CDRH2 selectedfrom the group consisting of SEQ ID NOs: 258, 278, 280, and 282, and aCDRH3 selected from the group consisting of SEQ ID NOs: 283, 285, 309,313, and 315, or a light chain amino acid sequence that comprises aCDRL1 selected from the group consisting of SEQ ID NOs: 320, 334, 337,and 340, a CDRL2 selected from the group consisting of SEQ ID NOs: 343,356, 351, and 344, and a CDRL3 selected from the group consisting of SEQID NOs: 360, 381, 385, and 387.

The first agent can be an antigen-binding protein that binds to HER-3and comprises a heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NOs: 42, 54, 70, 92, and 96. The antigen-bindingprotein can include a light chain amino acid sequence selected from thegroup consisting of SEQ ID NOs: 44, 56, 72, 94, and 98.

The first agent can be an antigen-binding protein that binds to HER-3and comprises a heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NOs: 42, 54, 70, 92, and 96; and a light chainamino acid sequence selected from the group consisting of SEQ ID NOs:44, 56, 72, 94, and 98.

The first agent can be an antigen-binding protein that binds to HER-3and comprises the heavy chain amino acid sequence of SEQ ID NO:42 andthe light chain amino acid sequence of SEQ ID NO:44. The first agent canbe an antigen-binding protein that binds to HER-3 and comprises theheavy chain amino acid sequence of SEQ ID NO:54 and the light chainamino acid sequence of SEQ ID NO:56. The first agent can be anantigen-binding protein that binds to HER-3 and comprises the heavychain amino acid sequence of SEQ ID NO:70 and the light chain amino acidsequence of SEQ ID NO:72. The first agent can be an antigen-bindingprotein that binds to HER-3 and comprises a CDRH3 selected from thegroup consisting of SEQ ID NOs: 283, 285, 309, 313, and 315. The firstagent can be an antigen-binding protein that binds to HER-3 andcomprises a CDHL3 selected from the group consisting of SEQ ID NOs: 360,381, 385, and 387.

The antigen-binding protein can be directed against the extracellulardomain of HER-3. Binding of the antigen-binding protein to HER-3 canreduce HER-3-mediated signal transduction, reduce HER-3 phosphorylation,reduce cell proliferation, reduce cell migration, and/or increase thedownregulation of HER-3.

The antigen-binding protein that binds to HER-3 can be an antibody. Theantibody can be a monoclonal antibody, a polyclonal antibody, arecombinant antibody, a humanized antibody, a human antibody, a chimericantibody, a multi-specific antibody, or an antibody fragment thereof(e.g., a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a Fvfragment, a diabody, or a single chain antibody molecule). The antibodycan be of the IgG1-, IgG2-, IgG3- or IgG4-type.

The first agent can be an antigen-binding protein that binds to HER-3,and the antigen-binding protein can be coupled to an effector group. Theeffector group can be a radioisotope or radionuclide, a toxin, or atherapeutic or chemotherapeutic group (e.g., a therapeutic orchemotherapeutic group selected from the group consisting ofcalicheamicin, auristatin-PE, geldanamycin, maytansine and derivativesthereof).

The second agent can be a small molecule compound or an antigen-bindingprotein. The second agent can be, for example, trastuzumab, lapatinib,neratinib, panitumumab, erlotinib, cetuximab, pertuzumab, and T-DM1.

In another aspect, this document features a method of treating orpreventing a disease associated with HER-3 in a subject, comprisingadministering to the subject a first agent and a second agent, whereinthe first agent is an antigen-binding protein that binds to HER-3 andcomprises the heavy chain amino acid sequence of SEQ ID NO:42 and thelight chain amino acid sequence of SEQ ID NO:44, and wherein the secondagent is selected from the group consisting of erlotinib, lapatinib, andneratinib. In addition, this document features methods of treating orpreventing a disease associated with HER-3 in a subject, comprisingadministering to the subject a first agent and a second agent, whereinthe first agent is an antigen-binding protein that binds to HER-3 andcomprises the heavy chain amino acid sequence of SEQ ID NO:54 and thelight chain amino acid sequence of SEQ ID NO:56, or an antigen-bindingprotein that binds to HER-3 and comprises the heavy chain amino acidsequence of SEQ ID NO:70 and the light chain amino acid sequence of SEQID NO:72, and wherein the second agent is selected from the groupconsisting of erlotinib, lapatinib, and neratinib.

This document also features a method of treating or preventing a diseaseassociated with HER-3 in a subject, comprising administering to thesubject a first agent and a second agent, wherein the first agent is anantigen-binding protein that binds to HER-3 and comprises the heavychain amino acid sequence of SEQ ID NO:42 and the light chain amino acidsequence of SEQ ID NO:44, and wherein the second agent is selected fromthe group consisting of trastuzumab, T-DM1, panitumumab, and cetuximab.

The methods provided herein can optionally include administering a thirdor further therapeutic agent and/or radiation therapy. The third orfurther therapeutic agent can be an anti-neoplastic agent (e.g., ananti-tumor antibody or a chemotherapeutic agent, such as capecitabine,anthracycline, doxorubicin, cyclophosphamide, paclitaxel, docetaxel,cisplatin, gemcitabine, or carboplatin).

The first agent and the second agent can be administered by intravenous,subcutaneous, intramuscular or oral administration. The disease can be ahyperproliferative disease (e.g., a disease selected from the groupconsisting of breast cancer, ovarian cancer, prostate cancer, coloncancer, renal cancer, lung cancer, pancreatic cancer, epidermoidcarcinoma, fibrosarcoma, melanoma, nasopharyngeal carcinoma, andsquamous cell carcinoma).

The methods provided herein can include administering the first agent ata dose of about 1 to about 20 mg/kg body weight, at least once every 6weeks. The methods can include administering the second agent at a doseof about 1 to about 20 mg/kg body weight, at least once every 6 weeks.The methods can further include, prior to the administering, using amethod that comprises analysis of a predictive marker to select asubject having a disease associated with HER-3. The methods can furtherinclude after the administering, monitoring the therapeutic outcome.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Thedisclosures of each of the publications, applications, patents, andother references mentioned herein are hereby incorporated herein byreference in their entireties. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph plotting the effects of a human anti-HER-3 antibodyand panitumumab, either alone or in combination, on non-small cell lungcancer (NSCLC) xenograft tumor (Calu-3) growth.

FIG. 2 is a graph plotting the effects of a human anti-HER-3 antibodyand erlotinib, either alone or in combination, on Calu-3 growth.

FIG. 3 is a graph plotting the effects of a human anti-HER-3 antibody,either alone or in combination with c2C4 (a HER2 dimerizationinhibitor), or trastuzumab on basal anchorage-independent growth ofSkBr-3 breast cancer cells.

FIG. 4 is a graph plotting the effects of a human anti-HER-3 antibody,either alone or in combination with c2C4, trastuzumab, or cetuximab, onHRG stimulated anchorage-independent growth of SkBr-3 breast cancercells.

FIG. 5 is a graph plotting the effects of a human anti-HER-3 antibody,either alone or in combination with 2C4), trastuzumab, or cetuximab, onbasal anchorage-independent growth of MDA-MB-435 ovarian cancer cells.

FIGS. 6A-6D are a series of graphs plotting the effects of a humananti-HER-3 antibody, either alone or in combination with trastuzumab(FIG. 6A), lapatinib (FIG. 6B), gemcitibine (FIG. 6C), or cisplatin(FIG. 6D), on proliferation of MDA-MB-175VII breast cancer cells.

FIG. 7 is a graph plotting the effects of a human anti-HER-3 antibody,either alone or in combination with c2C4, trastuzumab, or lapatinib, onHRG stimulated proliferation of ZR-75-30 breast cancer cells.

FIG. 8 is a graph plotting the effects of a human anti-HER-3 antibody,either alone or in combination with c2C4, trastuzumab, or lapatinib, onHRG stimulated proliferation of BT474 breast cancer cells.

FIG. 9 is a graph plotting the effects of a human anti-HER-3 antibody,either alone or in combination with cetuximab, c2C4, or trastuzumab, onproliferation of HRG stimulated DLD-1 colon cancer cells.

FIG. 10 is a graph plotting the effects of a human anti-HER-3 antibody,either alone or in combination with c2C4, or trastuzumab, or lapatinibon HRG stimulated proliferation of HCC-1569 breast cancer cells.

FIG. 11 is a graph plotting the effects of a human anti-HER-3 antibody,either alone or in combination with 2C4, trastuzumab, or lapatinib, onHRG stimulated proliferation of SkBr-3 breast cancer cells.

FIG. 12 is a graph plotting the effects of a human anti-HER-3 antibody,either alone or in combination with panitumumab on proliferation of FaDuhead and neck cancer cells.

FIG. 13 is a picture of a Western blot showing the effects of a humananti-HER-3 antibody, either alone or in combination with cetuximab,c2C4, or trastuzumab, on phosphorylation of HER-3 (top panel), Akt(middle panel), and ERK (bottom panel) in MDA-MB-175VII breast cancercells.

FIG. 14 is a picture of a Western blot showing the effects of a humananti-HER-3 antibody, either alone or in combination with cetuximab,c2C4, trastuzumab, or lapatinib, on phosphorylation of HER-3 (toppanel), Akt (middle panel), and ERK (bottom panel) in HRG stimulatedSkBr-3 breast cancer cells.

FIG. 15 is a picture of a Western blot showing the effects of a humananti-HER-3 antibody, either alone or in combination with cetuximab,pertuzumab (c2C4), or trastuzumab, on phosphorylation of HER-3 (toppanel) or Akt (bottom panel) in HRG stimulated Ls174T colon cancercells.

FIG. 16 is a picture of a Western blot showing the effects of a humananti-HER-3 antibody, either alone or in combination with cetuximab,c2C4, or trastuzumab, on phosphorylation of HER-3 (top panel), Akt(middle panel), and ERK (bottom panel) in HRG stimulated HCC 1569 breastcancer cells.

FIG. 17 is a picture of a Western blot showing the effects of a humananti-HER-3 antibody, either alone or in combination with panitumumab, onphosphorylation of Akt, PGFR, HER-2, HER-3, HER-4, and ERK in A549alveolar epithelial cells. Lane 1, IgG control; lane 2, panitumumab,alone; lane 3, U1-59, alone; lane 4, U1-59, in combination withpanitumumab. Tubulin was used as a control for equal loading.

FIG. 18 is a picture of a Western blot showing the effects of a humananti-HER-3 antibody, either alone or in combination with panitumumab orlapatinib, on phosphorylation of HER-3, Akt, HER-2, ERK, and EGF-R inCalu3 NSCLC cells. Lane 1, IgG control; lane 2, panitumumab alone; lane3, U1-59 alone; lane 4, lapatinib alone; lane 5, U1-59 in combinationwith panitumumab; lane 6, U1-59 in combination with lapatinib.

FIG. 19 is a graph plotting the effects of a human anti-HER-3 antibodyand lapatinib, either alone or in combination, on breast cancerxenograft tumor (HCC-1569) growth.

FIG. 20 shows that treatment of A549 NSCLC cells with U1-59 inhibitsHER3 phosphorylation and reduces reactivation after treatment withgefitinib. A549 cells were treated with gefitinib, U1-59 or both, andHER3 phosphorylation was evaluated by ELISA analysis. Treatment withgefitinib for 1 hour resulted in partial inhibition of HERphosphorylation, which was reversed to control levels after 24 hours. Incontrast, treatment with U1-59 led to greater inhibition of HERphosphorylation that was sustained after 24 hours. Combined treatmentwith both agents prevented the reversal of inhibition seen after 24hours in cells treated with gefitinib alone. Experiments were performedin triplicate wells and repeated at least 2 times. Results are expressedas mean±SD.

DETAILED DESCRIPTION

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

Generally, nomenclatures used in connection with, and techniques of celland tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present application are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2001), Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates (1992), and Harlow and LaneAntibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990), the disclosure of each reference ofwhich is hereby incorporated herein by reference in its entirety.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The terminology used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well known and commonly used in the art. Standardtechniques can be used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the disclosed, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean.+/−.1%.

1. General Overview

This document provides materials and methods related to treating orpreventing diseases associated with HER-3, using a combination of afirst agent that binds to HER-3, and a second agent that binds to/orinhibits the activity of other members of the HER family. The firstagent and the second agent may be a biological compound, such as anantigen binding protein, or a small molecular tyrosine kinase inhibitor.For example, provided herein are isolated polypeptides (e.g., bindingproteins such as antibodies), and/or small molecular tyrosine kinaseinhibitors that bind to and/or inhibit individual or multiple members ofthe HER family, such as HER-3, HER-2, EGF-R, HER-4, and/or any othermembers of the HER family. Also provided are compositions comprising afirst agent that binds to HER-3, and a second agent that binds to and/orinhibits the activity of one or multiple other HER family members, andmethods for using the same to treat or prevent HER-3 associated disease.

Certain first and/or second agents described herein are biologics, suchas antigen binding proteins. In certain embodiments, the polypeptidestructure of the antigen binding proteins is based on antibodies,including, but not limited to, monoclonal antibodies, bispecificantibodies, minibodies, domain antibodies, synthetic antibodies(sometimes referred to as “antibody mimetics”), chimeric antibodies,humanized antibodies, human antibodies, antibody fusions (sometimesreferred to as “antibody conjugates”), and fragments thereof,respectively. The various structures are further described below. Inother embodiments, the first and/or second agent is a small moleculartyrosine kinase inhibitor. In yet other embodiments, the first and/orsecond agent is an siRNA. In yet other embodiments, the first and/orsecond agent is a natural substance.

The compositions described herein, and the methods of using the same,have been demonstrated improved inhibition of the growth of solid tumorsthat express HER-3 and at least one other member of the HER family. Inparticular, administering a combination of a first agent that binds toHER-3 and a second agent that binds to and/or inhibits at least oneother member of the HER family has been demonstrated herein to haveincreased efficacy in inhibiting the growth of a variety of tumors, whencompared to the administration of either the first or the second agentalone. Therefore, the compositions and methods disclosed herein havedemonstrated utility in improved methods of treating and preventingneoplastic disease, such as cancer.

2. HER-3 Binding Agents

As described herein, the agent that binds to HER-3 can be a biologicalcompound, including, but not limited to, an antigen binding protein,such as an antibody, or a small molecular tyrosine kinase inhibitor. Asused herein, an “antigen binding protein” or “binding protein” as usedherein means a protein that specifically binds a specified targetantigen, such as member of the HER family, e.g., HER-3. An antigenbinding protein is said to “specifically bind” its target antigen whenthe dissociation constant (K_(D)) is ≦10⁻⁸ M. The antibody specificallybinds antigen with “high affinity” when the K_(D) is ≦5×10⁻⁹ M, and with“very high affinity” when the K_(D) is ≦5×10¹⁰ M. In one embodiment, theantibody has a K_(D) of ≦10⁻⁹ M and an off-rate of about 1×0.10⁻⁴/sec.In one embodiment, the off-rate is about 1×0.10⁵/sec. In otherembodiments, the antibodies will bind to a specified member of the HERfamily with a K_(D) of between about 10⁻⁸ M and 10⁻¹⁰ M, and in yetanother embodiment it will bind with a K_(D)≦2×0.10⁻¹⁰. Further, as usedherein, a small molecule compound is a low molecular weight compoundthat has been chemically synthesized to inhibit the enzymatic activityof one or more protein kinase, including serine, threonine or tyrosinekinases.

In some embodiments, where the HER-3 binding agent is a biologicalcompound, the agent is an antigen binding protein, such as an antibodythat can bind to HER-3. Thus provided herein for use in compositions andmethods of treating HER-3 associated diseases are HER binding proteins,including anti-HER-3 antibodies. In some embodiments, an antibodytargeted to HER-3 can be directed against the extracellular domain (ECD)of HER-3. For example, an anti-HER-3 antibody as described herein caninteract with at least one epitope in the extracellular part of HER-3.The epitopes can be located in the amino terminal L1 domain (aa 19-184),in the S1 (aa 185-327) and S2 (aa 500-632) cysteine-rich domains, in theL2 domain (328-499), which is flanked by the two cysteine-rich domains,or in a combination of HER-3 domains. The epitopes also may be locatedin combinations of domains such as, without limitation, an epitopecomprised by parts of L1 and S1.

A HER-3 binding protein can be further characterized in that its bindingto HER-3 reduces HER-3-mediated signal transduction. A reduction ofHER-3-mediated signal transduction may, e.g., be caused by adownregulation of HER-3 resulting in an at least partial disappearanceof HER-3 molecules from the cell surface or by a stabilization of HER-3on the cell surface in a substantially inactive form, i.e., a form thatexhibits a lower signal transduction compared to the non-stabilizedform. Alternatively, a reduction of HER-3-mediated signal transductionalso may be caused by influencing, e.g., decreasing or inhibiting, thebinding of a ligand or another member of the HER family to HER-3. Forexample, a reduction of HER-3 mediated signal transduction also can becaused by, decreasing the formation of HER-3 containing dimers withother HER family members (e.g., EGF-R).

A HER-3 binding agent can be a scaffold protein having an antibody-likebinding activity (e.g., having activity similar to an anti-HER-3antibody) or an antibody, i.e., an anti-HER-3 antibody. As used herein,the term “scaffold protein” means a polypeptide or protein with exposedsurface areas in which amino acid insertions, substitutions or deletionsare highly tolerable. Examples of scaffold proteins that can be used inaccordance with the present methods include protein A fromStaphylococcus aureus, the bilin binding protein from Pieris brassicaeor other lipocalins, ankyrin repeat proteins, and human fibronectin(reviewed in Binz and Plückthun (2005) Curr. Opin. Biotechnol.16:459-69). Engineering of a scaffold protein can be regarded asgrafting or integrating an affinity function onto or into the structuralframework of a stably folded protein. Affinity function means a proteinbinding affinity according to the present document. A scaffold can bestructurally separable from the amino acid sequences conferring bindingspecificity. In general, proteins appearing suitable for the developmentof such artificial affinity reagents may be obtained by rational, ormost commonly, combinatorial protein engineering techniques such aspanning against HER-3, either purified protein or protein displayed onthe cell surface, for binding agents in an artificial scaffold librarydisplayed in vitro, skills which are known in the art (see, e.g., Skerra(2000) J. Mol. Recog. 13:167-87; and Binz and Plückthun, supra). Inaddition, a scaffold protein having an antibody like binding activitycan be derived from an acceptor polypeptide containing the scaffolddomain, which can be grafted with binding domains of a donor polypeptideto confer the binding specificity of the donor polypeptide onto thescaffold domain containing the acceptor polypeptide. The insertedbinding domains may be, for example, the complementarity determiningregion (CDR) of an antibody, in particular an anti-HER-3 antibody.Insertion can be accomplished by various methods known to those skilledin the art including, for example, polypeptide synthesis, nucleic acidsynthesis of an encoding amino acid as well by various forms ofrecombinant methods well known to those skilled in the art.

The term “antibody” includes monoclonal antibodies, polyclonalantibodies, recombinant antibodies, humanized antibodies (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-329;and Presta (1992) Curr. Op. Struct. Biol. 2:593-596), chimericantibodies (Morrison et al. (1984) Proc. Natl. Acad. Sci. US81:6851-6855), multispecific antibodies (e.g., bispecific antibodies)formed from at least two antibodies, or antibody fragments thereof. Theterm “antibody fragment” comprises any portion of the afore-mentionedantibodies, such as their antigen binding or variable regions. Examplesof antibody fragments include Fab fragments, Fab′ fragments, F(ab′)₂fragments, Fv fragments, diabodies (Hollinger et al. (1993) Proc. Natl.Acad. Sci. US 90:6444-6448), single chain antibody molecules (Plückthunin: The Pharmacology of Monoclonal Antibodies 113, Rosenburg and Moore,eds., Springer Verlag, NY (1994), 269-315) and other fragments as longas they exhibit the desired capability of binding to HER-3.

In addition, the term “antibody,” as used herein, includes antibody-likemolecules that contain engineered sub-domains of antibodies or naturallyoccurring antibody variants. These antibody-like molecules may besingle-domain antibodies such as V_(H)-only or V_(L)-only domainsderived either from natural sources such as camelids (Muyldermans et al.(2001) Rev. Mol. Biotechnol. 74:277-302) or through in vitro display oflibraries from humans, camelids or other species (Holt et al. (2003)Trends Biotechnol. 21:484-90).

An “Fv fragment” is the minimum antibody fragment that contains acomplete antigen-recognition and -binding site. This region consists ofa dimer of one heavy chain variable domain and one light chain variabledomain in tight, non-covalent association. It is in this configurationthat the three CDR's of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six CDR's confer antigen-binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three CDR's specific for an antigen) has the ability torecognize and bind the antigen, although usually at a lower affinitythan the entire binding site. The “Fab fragment” also contains theconstant domain of the light chain and the first constant domain (CH1)of the heavy chain. The “Fab fragment” differs from the “Fab′ fragment”by the addition of a few residues at the carboxy terminus of the heavychain CH1 domain, including one or more cysteines from the antibodyhinge region. The “F(ab′)₂ fragment” originally is produced as a pair of“Fab′ fragments” which have hinge cysteines between them. Methods ofpreparing such antibody fragments, such as papain or pepsin digestion,are known to those skilled in the art.

An antibody can be of the IgA-, IgD-, IgE, IgG- or IgM-type, includingIgG- or IgM-types such as, without limitation, IgG1-, IgG2-, IgG3-,IgG4-, IgM1- and IgM2-types. For example, in some cases, the antibody isof the IgG1-, IgG2- or IgG4-type.

In certain respects, e.g., in connection with the generation ofantibodies as therapeutic candidates against HER-3, it may be desirablethat the antibody is capable of fixing complement and participating incomplement-dependent cytotoxicity (CDC). There are a number of isotypesof antibodies that are capable of the same including: murine IgM, murineIgG2a, murine IgG2b, murine IgG3, human IgM, human IgG1, human IgG3, andhuman IgA, for example. It will be appreciated that antibodies that aregenerated need not initially possess such an isotype but, rather theantibody as generated can possess any isotype and the antibody can beisotype switched by appending the molecularly cloned V region genes orcDNA to molecularly cloned constant region genes or cDNAs in appropriateexpression vectors using conventional molecular biological techniquesthat are well known in the art and then expressing the antibodies inhost cells using techniques known in the art. The isotype-switchedantibody may also possess an Fc region that has been molecularlyengineered to possess superior CDC over naturally occurring variants(Idusogie et al. (2001) J. Immunol. 166:2571-2575) and expressedrecombinantly in host cells using techniques known in the art. Suchtechniques include the use of direct recombinant techniques (see, e.g.,U.S. Pat. No. 4,816,397), cell-cell fusion techniques (see, e.g., U.S.Pat. Nos. 5,916,771 and 6,207,418), among others. In the cell-cellfusion technique, a myeloma or other cell line such as CHO is preparedthat possesses a heavy chain with any desired isotype and anothermyeloma or other cell line such as CHO is prepared that possesses thelight chain. Such cells can thereafter be fused, and a cell lineexpressing an intact antibody can be isolated. By way of example, ahuman anti-HER-3 IgG4 antibody that possesses the desired binding to theHER-3 antigen can be readily isotype switched to generate a human IgM,human IgG1 or human IgG3 isotype, while still possessing the samevariable region (which defines the antibody's specificity and some ofits affinity). Such a molecule might then be capable of fixingcomplement and participating in CDC.

Moreover, an antibody also may be capable of binding to Fc receptors oneffector cells such as monocytes and natural killer (NK) cells, andparticipating in antibody-dependent cellular cytotoxicity (ADCC). Thereare a number of antibody isotypes that are capable of the same,including, without limitation, the following: murine IgG2a, murineIgG2b, murine IgG3, human IgG1 and human IgG3. It will be appreciatedthat the antibodies that are generated need not initially possess suchan isotype but, rather the antibody as generated can possess any isotypeand the antibody can be isotype switched by appending the molecularlycloned V region genes or cDNA to molecularly cloned constant regiongenes or cDNAs in appropriate expression vectors using conventionalmolecular biological techniques that are well known in the art and thenexpressing the antibodies in host cells using techniques known in theart. The isotype-switched antibody may also possess an Fc region thathas been molecularly engineered to possess superior ADCC over naturallyoccurring variants (Shields et al. (2001) J. Biol. Chem. 276:6591-604)and expressed recombinantly in host cells using techniques known in theart. Such techniques include the use of direct recombinant techniques(see, e.g., U.S. Pat. No. 4,816,397), cell-cell fusion techniques (see,e.g., U.S. Pat. Nos. 5,916,771 and 6,207,418), among others. In thecell-cell fusion technique, a myeloma or other cell line such as CHO isprepared that possesses a heavy chain with any desired isotype andanother myeloma or other cell line such as CHO is prepared thatpossesses the light chain. Such cells can thereafter be fused, and acell line expressing an intact antibody can be isolated. By way ofexample, a human anti-HER-3 IgG4 antibody that possesses the desiredbinding to the HER-3 antigen could be readily isotype switched togenerate a human IgG1 or human IgG3 isotype, while still possessing thesame variable region (which defines the antibody's specificity and someof its affinity). Such molecule might then be capable of binding to FcγRon effectors cells and participating in ADCC.

TABLE 10 herein provides amino acid sequences for a number of CDR's thatcan be included in antibodies against HER-3. In some embodiments, anisolated binding protein targeted to HER-3 can include a heavy chainamino acid sequence containing at least one CDR selected from the groupconsisting of: (a) CDRH1's as shown in SEQ ID NOS:2, 6, 10, 14, 18, 22,26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88,92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146,150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202,206, 210, 214, 218, 222, 226 and 230, (b) CDRH2's as shown in SEQ IDNOS:2, 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62,66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122,126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178,182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226 and 230, and(c) CDRH3's as shown in SEQ ID NOS:2, 6, 10, 14, 18, 22, 26, 30, 34, 36,40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100,104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 154,158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210,214, 218, 222, 226 and 230, and/or a light chain amino acid sequencecomprising at least one of the CDR's selected from the group consistingof: (d) CDRL1's as shown in SEQ ID NOS:4, 8, 12, 16, 20, 24, 28, 32, 38,44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110,114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168,172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224,228 and 232, (c) CDRL2's as shown in SEQ ID NOS:4, 8, 12, 16, 20, 24,28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102,106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160,164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216,220, 224, 228 and 232, and (f) CDRL3's as shown in SEQ ID NOS:4, 8, 12,16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90,94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152,156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208,212, 216, 220, 224, 228 and 232, as shown in the sequence listing filedherewith.

In some embodiments, an isolated binding protein targeted to HER-3 caninclude a heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NOS:2, 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42,46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108,112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162,166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218,222, 226 and 230, and/or a light chain amino acid sequence selected fromthe group consisting of SEQ ID NOS:4, 8, 12, 16, 20, 24, 28, 32, 38, 44,48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114,118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172,176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228 and232, as shown in the sequence listing filed herewith.

In some embodiments, an anti-HER-3 antibody can include a heavy chainamino acid sequence and a light chain amino acid sequence as shown inSEQ ID NOS:2 and 4, 6 and 8, 10 and 12, 14 and 16, 18 and 20, 22 and 24,26 and 28, 30 and 32, 36 and 38, 42 and 44, 46 and 48, 50 and 52, 54 and56, 60 and 58, 62 and 64, 66 and 68, 70 and 72, 74 and 76, 78 and 82, 80and 82, 84 and 86, 88 and 90, 92 and 94, 96 and 98, 100 and 102, 104 and106, 108 and 110, 112 and 114, 116 and 118, 122 and 124, 126 and 128,130 and 132, 134 and 136, 138 and 140, 142 and 144, 146 and 148, 150 and152, 154 and 156, 158 and 160, 162 and 164, 166 and 168, 170 and 172,174 and 176, 178 and 180, 182 and 184, 186 and 188, 190 and 192, 194 and196, 198 and 200, 202 and 204, 206 and 208, 210 and 212, 214 and 216,218 and 220, 222 and 224, 226 and 228, 230 and 232, or a heavy chainamino acid sequence as shown in any one of SEQ ID NOS:34, 40, 60, 62,and 120, or a light chain amino acid sequence as shown in either of SEQID NOS: 58 and 64, as shown in the sequence listing filed herewith.

In some embodiments, a protein targeted to HER-3 can be a scaffoldprotein having an antibody-like binding activity (e.g., having activitysimilar to an anti-HER-3 antibody), or an antibody, e.g., an anti-HER-3antibody. The anti-HER-3 antibody can be selected from the groupconsisting of antibodies designated U1-1, U1-2, U1-3, U1-4, U1-5, U1-6,U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13, U1-14, U1-15, U1-16,U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23, U1-24, U1-25, U1-26,U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33, U1-34, U1-35, U1-36,U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43, U1-44, U1-45, U1-46,U1-47, U1-48, U1-49, U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55,U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, and U1-62, or an antibodyhaving at least one heavy or light chain of one of the aforesaidantibodies. The antibodies designated as U1-49 (SEQ ID NO: 42/44), U1-53(SEQ ID NO: 54/56), and U1-59 (SEQ ID NO: 70/72), or an antibody havingat least one heavy or light chain of one of these antibodies, can beparticularly useful.

It is to be understood that the amino acid sequence of the HER-3 bindingproteins provided herein is not limited to the twenty conventional aminoacids (see, Immunology—A Synthesis (2^(nd) Edition, Golub and Gren,eds., Sinauer Associates, Sunderland, Mass. (1991), the disclosure ofwhich is hereby incorporated herein by reference in its entirety). Forexample, the amino acids may include stereoisomers (e.g., D-amino acids)of the twenty conventional amino acids, unnatural amino acids such asα-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, andother unconventional amino acids. Examples of unconventional aminoacids, which may also be suitable components for the binding proteinsprovided herein, include: 4-hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids, e.g.,4-hydroxyproline.

Furthermore, minor variations in the amino acid sequences shown in SEQID NOS:1-390 (as set forth in the appendix filed herewith) arecontemplated as being encompassed by the present disclosure, providedthat the variations in the amino acid sequence maintain at least 75%(e.g., at least 80%, 90%, 95%, or 99%) of the sequences shown in SEQ IDNOS:1-390. Variations can occur within the framework regions (i.e.,outside the CDRs), within the CDRs, or within the framework regions andthe CDRs. In some embodiments, variations in the amino acid sequencesshown in SEQ ID NOS:1-390, i.e., deletions, insertions and/orsubstitutions of at least one amino acid, can occur near boundaries offunctional domains. Structural and functional domains can be identifiedby comparison of the nucleotide and/or amino acid sequence data topublic or proprietary sequence databases. Computerized comparisonmethods can be used to identify sequence motifs or predicted proteinconformation domains that occur in other binding proteins of knownstructure and/or function. Methods for identifying protein sequencesthat fold into a known three-dimensional structure are known in the art.(See, e.g., Bowie et al. (1991) Science 253:164; Proteins, Structuresand Molecular Principles, Creighton, Ed., W H. Freeman and Company, NewYork (1984); Introduction to Protein Structure, Branden and Tooze, eds.,Garland Publishing, New York, N.Y. (1991); and Thornton et al. (1991)Nature 354:105, the disclosure of each reference of which is herebyincorporated herein by reference in its entirety.) Thus, those of skillin the art can recognize sequence motifs and structural conformationsthat may be used to define structural and functional domains inaccordance with the proteins described herein.

Variations in the amino acid sequences shown in SEQ ID NOS:1-390 caninclude those that lead to a reduced susceptibility to proteolysis oroxidation, alter glycosylation patterns or alter binding affinities orconfer or modify other physicochemical or functional properties of thebinding protein. In particular, conservative amino acid replacements arecontemplated. Conservative replacements are those that take place withina family of amino acids that are related in their side chains. Aminoacid families include the following: acidic family=aspartate, glutamate;basic family=lysine, arginine, histidine; non-polar family=alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan; and uncharged polar family=glycine, asparagine, glutamine,cysteine, serine, threonine, tyrosine. Alternative families include:aliphatic-hydroxy family=serine and threonine; amide-containingfamily=asparagine and glutamine; aliphatic family=alanine, valine,leucine and isoleucine; and aromatic family=phenylalanine, tryptophan,and tyrosine. For example, it is reasonable to expect that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid will not have a majoreffect on the binding or properties of the resulting binding protein,especially if the replacement does not involve an amino acid within aframework site. However, all other possible amino acid replacements alsoare encompassed herein. Whether an amino acid change results in afunctional HER-3 binding protein that reduces signal transduction ofHER-3 can readily be determined by assaying the specific HER-3 bindingactivity of the resulting binding protein by ELISA or FACS, or in vitroor in vivo functional assays.

In some embodiments, a HER-3 binding protein can be coupled to aneffector group. Such a binding protein can be especially useful fortherapeutic applications. As used herein, the term “effector group”refers to a cytotoxic group such as a radioisotope or radionuclide, atoxin, a therapeutic group or other effector group known in the art.Examples of suitable effector groups are radioisotopes or radionuclides(e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I) or non-radioisotopes (e.g., 2D), calicheamicin, dolastatin analogs such asauristatins, and chemotherapeutic agents such as geldanamycin andmaytansine derivates, including DM1. Thus, in some cases, a group can beboth a labeling group and an effector group. Various methods ofattaching effector groups to polypeptides or glycopolypeptides (such asantibodies) are known in the art, and may be used in making and carryingout the compositions and methods described herein. In some embodiments,it may be useful to have effector groups attached to a binding proteinby spacer arms of various lengths to, for example, reduce potentialsteric hindrance.

This document also relates to processes for preparing an isolated HER-3binding protein, comprising the step of preparing the protein from ahost cell that expresses the protein. Host cells that can be usedinclude, without limitation, hybridomas, eukaryotic cells (e.g.,mammalian cells such as hamster, rabbit, rat, pig, or mouse cells),plant cells, fungal cells, yeast cells (e.g., Saccharomyces cerevisiaeor Pichia pastoris cells), prokaryotic cells (e.g., E. coli cells), andother cells used for production of binding proteins. Various methods forpreparing and isolating binding proteins, such as scaffold proteins orantibodies, from host cells are known in the art and may be used inperforming the methods described herein. Moreover, methods for preparingbinding protein fragments, e.g., scaffold protein fragments or antibodyfragments, such as papain or pepsin digestion, modern cloningtechniques, techniques for preparing single chain antibody molecules(Plückthun, supra) and diabodies (Hollinger et al., supra), also arcknown to those skilled in the art and may be used in performing thepresently described methods.

In some embodiments, a HER-3 binding protein can be prepared from ahybridoma that secretes the protein. See, e.g., Köhler et al. (1975)Nature 256:495.

In some embodiments, a HER-3 binding protein can be preparedrecombinantly by optimizing and/or amplifying expression of the bindingprotein in host cells, and isolating the binding protein from the hostcells. To this end, host cells can be transformed or transfected withDNA (e.g., a vector) encoding a HER-3 binding protein, and culturedunder conditions appropriate to produce the binding protein. See, e.g.,U.S. Pat. No. 4,816,567. Useful host cells include, for example, CHOcells, NS/0 myeloma cells, human embryonic kidney 293 cells, E. colicells, and Saccharomyces cerevisiae cells.

HER-3 binding proteins that are antibodies can be prepared from animalsgenetically engineered to make fully human antibodies, or from anantibody display library made in bacteriophage, yeast, ribosome or E.coli. See, e.g., Clackson et al. (1991) Nature 352:624-628; Marks et al.(1991) J. Mol. Biol. 222:581-597; Feldhaus and Siegel (2004) J. Immunol.Methods 290:69-80; Groves and Osbourn (2005) Expert Opin. Biol. Ther.5:125-135; and Jostock and Dubel (2005) Comb. Chem. High ThroughputScreen 8:127-133.

In some embodiments, antibodies as provided herein can be fully human orhumanized antibodies. Human antibodies avoid certain problems associatedwith xenogeneic antibodies, such as antibodies that possess murine orrat variable and/or constant regions. The presence of xenogeneic-derivedproteins can lead to an immune response against the antibody by apatient, subsequently leading to the rapid clearance of the antibody,loss of therapeutic utility through neutralization of the antibody,and/or severe, even life-threatening, allergic reactions. To avoid theusing murine or rat-derived antibodies, fully human antibodies can begenerated through the introduction of functional human antibody lociinto a rodent or another mammal or animal so that the rodent, othermammal or animal produces fully human antibodies.

One method for generating fully human antibodies is to utilizeXENOMOUSE® strains of mice that have been engineered to contain 245 kband 190 kb-sized germline configuration fragments of the human heavychain locus and kappa light chain locus. Other XENOMOUSE® strains ofmice contain 980 kb and 800 kb-sized germline configuration fragments ofthe human heavy chain locus and kappa light chain locus. Still otherXENOMOUSE® strains of mice contain 980 kb and 800 kb-sized germlineconfiguration fragments of the human heavy chain locus and kappa lightchain locus plus a 740 kb-sized germline configured complete humanlambda light chain locus. See, Mendez et al. (1997) Nature Genetics15:146-156; and Green and Jakobovits (1998) J. Exp. Med. 188:483-495.XENOMOUSE® strains are available from Amgen, Thousand Oaks, Calif.

The production of XENOMOUSE® mice is further discussed and delineated inUS Patent Publication 2003/0217373, filed Nov. 20, 2002; U.S. Pat. Nos.5,939,598, 6,075,181, 6,114,598, 6,150,584, 6,162,963, 6,673,986,6,833,268, and 7,435,871, and Japanese Patent Nos. 3068180B2, 3068506B2,and 3068507B2. See, also, European Patent No. EP0463151, PCT PublicationNos. WO 94/02602, WO 96/34096, WO 98/24893, and WO 00/76310. Thedisclosures of each of the above-cited patents, applications, andreferences is hereby incorporated herein by reference in its entirety.

Alternatively, a “minilocus” approach can be used. In the minilocusapproach, an exogenous Ig locus is mimicked through the inclusion ofpieces (individual genes) from the Ig locus. Thus, one or more V_(H)genes, one or more D_(H) genes, one or more J_(H) genes, a mu constantregion, and a second constant region (e.g., a gamma constant region) areformed into a construct for insertion into an animal. This approach isdescribed in U.S. Pat. Nos. 5,545,806, 5,545,807, 5,569,825, 5,591,669,5,612,205, 5,625,126, 5,625,825, 5,633,425, 5,643,763, 5,661,016,5,721,367, 5,770,429, 5,789,215, 5,789,650, 5,814,318, 5,874,299,5,877,397, 5,981,175, 6,023,010, 6,255,458, the disclosures of which arehereby incorporated herein by reference in their entireties. See, also,EP Patent No. 0546073, and PCT Publication Nos. WO 92/03918, WO92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO94/25585, WO 96/14436, WO 97/13852, and WO 98/24884, the disclosures ofwhich are hereby incorporated herein by reference in their entireties.

Human antibodies also can be generated from mice in which, throughmicrocell fusion, large pieces of chromosomes, or entire chromosomes,have been introduced. See, EP Patent Application Nos. 773288 and 843961,the disclosures of which are hereby incorporated herein by reference intheir entireties. Additionally, KM™ mice, which are the result ofcross-breeding of Kirin's Tc mice with Medarex's minilocus (Humab) micehave been generated. These mice possess the HC transchromosome of theKirin mice and the kappa chain transgene of the Medarex mice (Ishida etal. (2002) Cloning Stem Cells 4:91-102).

Human antibodies also can be derived by in vitro methods. Suitableexamples include, but are not limited to, phage display (ascommercialized by Cambridge Antibody Technology, Morphosys, Dyax,Biosite/Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), andAffimed), ribosome display (as commercialized by Cambridge AntibodyTechnology), yeast display, and the like.

As described herein, antibodies were prepared using XENOMOUSE®technology, as described below. Such mice are capable of producing humanimmunoglobulin molecules and antibodies, and are deficient in theproduction of murine immunoglobulin molecules and antibodies.Technologies utilized for achieving the same are disclosed in thepatents, applications, and references disclosed herein. For example,transgenic production of mice and antibodies therefrom is disclosed inU.S. patent application Ser. No. 08/759,620, filed Dec. 3, 1996, and PCTPublication Nos. WO 98/24893 and WO 00/76310, the disclosures of whichare hereby incorporated herein by reference in their entireties. Seealso Mendez et al. (1997) Nature Genetics 15:146-156, the disclosure ofwhich is hereby incorporated herein by reference in its entirety.

Using technology as described herein, fully human monoclonal antibodiesto a variety of antigens can be produced. For example, XENOMOUSE® linesof mice can be immunized with a HER-3 antigen of interest (e.g., HER-3or a fragment thereof), lymphatic cells (such as B-cells) can berecovered from mice that express antibodies, and the recovered celllines can be fused with a myeloid-type cell line to prepare immortalhybridoma cell lines. These hybridoma cell lines can be screened andselected to identify hybridoma cell lines that produce antibodiesspecific to the antigen of interest. Provided herein are methods for theproduction of multiple hybridoma cell lines that produce antibodiesspecific to HER-3. Further provided herein are methods forcharacterizing antibodies produced by such cell lines, includingnucleotide and amino acid sequence analyses of the heavy and lightchains of such antibodies.

In general, antibodies produced by fused hybridomas as described beloware human IgG1 heavy chains with fully human kappa light chains,although some antibodies described herein possess human IgG4 heavychains as well as IgG1 heavy chains. Antibodies also can be of otherhuman isotypes, including IgG2 and IgG3. The antibodies generally havehigh affinities, with a K_(D) typically from about 10⁻⁶ to about 10⁻¹³ Mor below, when measured by solid phase and cell-based techniques.

This document also provides isolated nucleic acid molecules that encodeHER-3 binding proteins as described herein. The term “isolated nucleicacid molecule,” as used herein, refers to a polynucleotide of genomic,cDNA, or synthetic origin, or some combination thereof, which (1) is notassociated with all or a portion of a polynucleotide with which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide to which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence. Further, the term “nucleicacid molecule,” as used herein, means a polymeric form of nucleotides ofat least 10 bases in length, either ribonucleotides or deoxynucleotidesor a modified form of either type of nucleotide, such as nucleotideswith modified or substituted sugar groups and the like. The term alsoincludes single and double stranded forms of DNA.

In some embodiments, a nucleic acid molecule can be operably linked to acontrol sequence. The term “control sequence,” as used herein, refers topolynucleotide sequences that are necessary to effect the expression andprocessing of coding sequences to which they are ligated. The nature ofsuch control sequences differs depending upon the host organism. Inprokaryotes, such control sequences generally include promoters,ribosomal binding sites, and transcription termination sequences. Ineukaryotes, generally, such control sequences include promoters andtranscription termination sequences. The term “control sequence” isintended to include, at a minimum, all components whose presence isessential for expression and processing, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Furthermore, the term “operably linked”,as used herein, refers to positions of components so described which arein a relationship permitting them to function in their intended manner.Moreover, an expression control sequence operably linked to a codingsequence is ligated in such a way that expression of the coding sequenceis achieved under conditions compatible with the expression controlsequence.

Also provided herein are vectors comprising a nucleic acid moleculeencoding a binding protein as disclosed herein. The nucleic acidmolecule can be operably linked to a control sequence. Furthermore, thevector may additionally contain a replication origin or a selectionmarker gene. Examples of vectors that may be used include, e.g.,plasmids, cosmids, phages, and viruses.

This document also provides host cells transformed with a nucleic acidmolecule or vector as described herein. Transformation can beaccomplished by any known method for introducing polynucleotides into ahost cell, including, for example, packaging the polynucleotide in avirus (or into a viral vector) and transducing a host cell with thevirus (or vector), or by transfection procedures known in the art, asexemplified by U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and4,959,455, the disclosures of which are hereby incorporated herein byreference in their entireties. Methods for introducing heterologouspolynucleotides into mammalian cells are well known in the art, andinclude, without limitation, dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, protoplastfusion, electroporation, encapsulation of the polynucleotide(s) inliposomes, and direct microinjection of the DNA into nuclei. Examples ofhost cells that may be used include hybridomas, eukaryotic cells (e.g.,mammalian cells such as hamster, rabbit, rat, pig, mouse, or otheranimal cells), plant cells (e.g., corn and tobacco cells), fungal cells(e.g., S. cerevisiae and P. pastoris cells), prokaryotic cells such asE. coli, and other cells used in the art for production of antibodies.Mammalian cell lines available as hosts for expression are well known inthe art and include, for example, many immortalized cell lines availablefrom the American Type Culture Collection (ATCC; Manassas, Va.). Theseinclude, without limitation, Chinese hamster ovary (CHO) cells, HeLacells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2 cells), and a number ofother cell lines.

In other embodiments, the agent binding to HER-3 is a small moleculecompound. Such compounds can be identified using, for example, physicalor virtual libraries of small molecules. In some embodiments, forexample, useful small molecule compounds can be identified usingconsensus virtual screening methods based on known HER-3 inhibitors andmodels of HER-3 active and inactive state structures. Compounds thatappear to be of interest can be further analyzed for structural noveltyand desirable physicochemical properties. Candidate compounds identifiedby virtual screening can be tested in vitro for, e.g., the ability toinhibit growth of cells that overexpress HER-3. In other embodiments,useful small molecule compounds can be identified from a library ofsmall molecule compounds, using high throughput methods to screen largenumbers of compounds for the ability to bind to and/or inhibit activityof HER-3 (e.g., in cells that overexpress HER-3). Small molecule HER-3inhibitors can be synthesized using standard chemical synthesis methods,for example.

In yet anther embodiment, the agent that binds to HER-3 may be a siRNAthat interferes with the expression of HER-3. An example of siRNA isEZN-3920 (antisense targeting erbB3 mRNA) (Santaris Pharma, Hoersholm,Denmark).

In yet other embodiments, the agent that binds HER-3 may be a naturalsubstance. For example, Kahalalide F, a marine-derived agent, has beensuggested to inhibit HER-3 oncogenic signaling (Jimeno et al. (2006) J.Translational Med. 4:3) by down-regulating HER-3 protein expression andAKT signaling (Janmaat et al. (2005) Mol. Pharmacol. 68:502-510).

In further embodiments, the agent that binds HRE-3 may be an artificialor naturally-occurring scaffold which is not an anti-HER-3 antibody, buthas an antibody-like activity (e.g., has an activity similar to that ofan anti-HER-3 antibody).”

3. Agents that Bind to Other HER Family Members

As outlined above, the compositions and methods provided herein fortreatment of HER-3 associated disease include a first agent that bindsto HER-3, in combination with a second agent that binds and/or inhibitsat least one other member of the HER family, including but not limitedto, EGF-R, HER-2, HER-4. The second agent can be, without limitation,biological drug, e.g., a binding protein, such as an antibodyspecifically binding to a member of the HER family, a small molecularcompound that binds to and/or alters (e.g., inhibits) the activity of atleast one member of the HER family other than (or in addition to) HER-3,an siRNA, or a natural substance. As used herein, the terms “other HERfamily members” and “another HER family member” refer to HER familymembers that are not HER-3. Examples are the EGF-R, HER-2, and HER-4,but “HER family member” also includes family members that have not yetbeen identified.

The second agent can alter the activity (e.g., increase or decrease) theactivity of the other HER family member, either through a direct effector an indirect effect on the HER family member. It is noted, however,that all second agents as provided herein will have an effect on HERfamily function and activity.

In some cases, for example, the second agent can be an antibody that canbind to another HER family member (e.g., EGF-R, HER-2, or HER-4), or toanother molecule that in turn can affect the activity of the other HERfamily member. Such an antibody can be targeted, for example, to theextracellular domain of the other HER family member, or to any othersuitable domain thereof (e.g., a kinase domain or a dimerizationdomain).

A second agent can be further characterized in that its effect onanother HER family member reduces HER-mediated signal transduction. Areduction of HER-mediated signal transduction may, e.g., be caused bydownregulation of the targeted HER family member, resulting in an atleast partial disappearance of the HER molecule from the cell, or by astabilization of the HER family member in a substantially inactive form.Alternatively, a reduction of HER-mediated signal transduction may becaused by influencing, e.g., decreasing or inhibiting, the binding of aligand to the HER family member, the binding of the HER family member toHER-3, or the binding of GRB2 to HER-2 or GRB2 to SHC, or, by inhibitingreceptor tyrosine phosphorylation, AKT phosphorylation, PYK2 tyrosinephosphorylation, or ERK2 phosphorylation, or any other cellularcomponent affecting the HER-family mediated signal transduction pathway.For example, a reduction of HER mediated signal transduction can becaused by decreasing the formation of dimers containing HER-3 andanother HER family member (e.g., EGF-R, HER-2, or HER-4). Regardless ofthe mechanism behind the function, it is noted that the second agent canserve to amplify the effect of the first agent that is targeted toHER-3.

In some embodiments, an agent that binds to another HER family member oranother protein that in turn affects activity of another HER familymember can be a scaffold protein having an antibody like bindingactivity (e.g., having activity similar to an anti-HER-3 antibody) or anantibody (e.g., an anti-EGF-R, anti-HER-2, or anti-HER-4 antibody).Scaffold proteins and antibodies in this context are as defined anddescribed above for agents targeted to HER-3. Such scaffold can beartificial or naturally-occurring.

It is noted, in some embodiments, the first agent that binds to HER-3,and the second agent that binds to and/or inhibits another HER familymember are combined within one compound, such as a bispecific antibody.

Also as described above, the amino acid sequences of proteins that bindto other HER family members, or to other proteins that in turn affectthe activity of another HER family member, are not limited to the twentyconventional amino acids. Further, as for the HER-3 binding proteinsdescribed herein, an agent that binds to or otherwise affects theactivity of another HER family member can be coupled to an effectorgroup.

This document also relates to processes for preparing isolated proteins(e.g., antibodies) that can bind to other HER family members, forexample. Such processes include those described above in the context ofHER-3 binding proteins. In some embodiments, antibodies (e.g., anti-HER,anti-HER-2, or anti-HER-4 antibodies, respectively) can be prepared fromanimals engineered to make fully human antibodies, or from an antibodydisplay library made in bacteriophage, yeast, ribosomes, or E. coli.Further, an antibody targeted directly or indirectly to another HERfamily member can be fully human or humanized, as described above.

Also provided herein are isolated nucleic acid molecules (e.g., vectors)expressing proteins that can bind to other HER family members and otherproteins that can affect the activity of other HER family members.Protein coding sequences within such nucleic acid molecules can beoperably linked to one or more control sequences, as described above.Further, nucleic acid molecules can be transformed or transfected into ahost cell as described above.

In some embodiments, the second agent is a small molecular tyrosinekinase inhibitor provided that the agent can affect (either directly orindirectly) the activity of a HER family member other than (or inaddition to) HER-3. Such inhibitors can be identified using, forexample, physical or virtual libraries of small molecules. In someembodiments, for example, useful small molecule compounds can beidentified using consensus virtual screening methods based on knowntyrosine kinase inhibitors and models of HER family member structures inactive and inactive states. Compounds that are initially identified asbeing of potential interest can be further analyzed for structuralnovelty and desirable physicochemical properties. Candidate compoundsidentified by virtual screening can be tested in vitro for, e.g., theability to inhibit growth of cells that overexpress a HER family memberother than HER-3. In other embodiments, useful small molecule tyrosinekinase inhibitors can be identified from a library of small moleculecompounds and using high throughput methods to screen large numbers ofthe compounds for the ability to bind to and/or inhibit activity of oneor more HER family members other than HER-3 (e.g., in cells thatoverexpress the HER protein). Small molecular tyrosine kinase inhibitorscan be synthesized using, for example, standard chemical synthesismethods.

Agents that can affect an activity of EGF-R (HER) include AEE-788(Novartis, Basel, Switzerland),BIBW-2992(N-[4-(3-chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-2-butenamide(Boehringer Ingelheim, Ingelheim, Germany), BMS-599626 (Bristol-MyersSquibb, New York, N.Y.), BMS-690514 (Bristol-Myers Squibb, New York,N.Y.), carnetinib dihydrochloride(N-[4-[N-(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]quinazolin-6-yl]acrylamidedihydrochloride (Pfizer, New York, N.Y.), CNX-222 (Avila Therapeutics,Waltham, Mass.), CUDC-101 (Curis, U.S. Pat. No. 7,547,781), Dimercept(Receptor Biologix, Palo Alto, Calif.), lapatinib (ditosilate hydrate(N-[3-chloro-4-[(3-fluorobenzyl)oxy]phenyl]-6-[5-[[[2-(methylsulfonyl)ethyl]amino]methyl]furan-2-yl]quinazolin-4-aminebis(4-methylbenzene-sulfonate)monohydrate (GlaxoSmithKline, London,England), MP-412 (Mitsubishi Tanabe Pharma Co., Osaka, Japan), neratinib((2E)-N-[4-[[3-chloro-4-[(pyridin-2-yl)methoxy]phenyl]]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide)(Wyeth, Madison, N.J.), S-222611 (Shionogi, Osaka, Japan), varlitinib(4-N-[3-chloro-4-(thiazol-2-ylmethoxy)phenyl]-6-N-[(4R)-4-methyl-4,5-dihydrooxazol-2-yl]quinazoline-4,6-diaminebis(4-methylbenzenesulfonate) (Array BioPharma, Boulder, Colo.),AGT-2000 (ArmeGen Technologies, Santa Monica, Calif.), AZD-4769(AstraZeneca, London, England), BIBX-1382 (Boehringer Ingelheim,Ingelheim, Germany), CGP-52411(4,5-bis(phenylamino)-1H-isoindole-1,3(2H)-dione) (Novartis, Basel,Switzerland), CL-387785(N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide) (Wyeth,Madison, N.J.), CP-292597 (Pfizer, New York, N.Y.), DAB-1059 (MitsubishiTanabe Pharma Co., Osaka, Japan), erlotinib(hydrochloride(4-(3-ethynylphenylamino)-6,7-bis(2-methoxyethoxy)-quinazolinehydrochloride (OSI Pharmaceuticals, Long Island, N.Y., U.S. Pat. No.5,747,498), gefitinib(4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxy]quinazoline)(AstraZeneca, London, England, U.S. Pat. No. 5,821,246), HMPL-813(Hutchison China MediTech, Hong Kong), MDP-01, (Med Discovery,Plan-Les-Ouates, Switzerland), MT-062 (Medisyn Technologies,Minneapolis, Minn.), ONC-101 (Oncalis, Schlieren, Switzerland),PD-153035, (4-(3-bromophenylamino)-6,7-dimethoxyquinazoline)(AstraZeneca, London, England), PD-169540 (Pfizer, New York, N.Y.),pelitinib (Wyeth Pharmaceuticals, Madison, N.J.), PF-299804 (Pfizer, NewYork, N.Y.), PKI-166(4-(R)-phenethylamino-6-(hydroxyl)phenyl-7H-pyrrolo[2.3-d]-pyrimidine)(Novartis, Basel, Switzerland), vandetanib(N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine)(AstraZeneca, London, England), VGA-1102 (Taisho Pharmaceuticals, Tokyo,Japan), WHI-P154(4-(3′-bromo-4′-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline), ZD-1838(AstraZeneca, London, England), cetuximab (ImClone Systems, New York,N.Y.), panitumumab (Amgen, Thousand Oaks, Calif.).

Agents that can affect an activity of HER2 include AEE-788 (Novartis,Basel, Switzerland), ARRY-333786 (Array BioPharma, Boulder, Colo.),ARRY-380 (Array BioPharma, Boulder, Colo.), BIBW-2992(N-[4-(3-chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-2-butenamide(Boehringer Ingelheim, Ingelheim, Germany), BMS-599626 (Bristol-MyersSquibb, New York, N.Y.), BMS-690514 (Bristol-Myers Squibb, New York,N.Y.), carnetinib dihydrochloride(N-[4-[N-(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]quinazolin-6-yl]acrylamidedihydrochloride) (Pfizer, New York, N.Y.), CNF-201 (Biogen Idec, SanDiego, Calif.), CNX-222 (Avila Therapeutics, Waltham, Mass.), CP-654577(OSI Pharmaceuticals, Long Island, N.Y.), CP-724714(2-methoxy-N-[3-[4-[3-methyl-4-(6-methyl-pyridin-3-yloxy)phenyl-amino]quinazolin-6-yl]-E-allyl]acetamide)(OSI Pharmaceuticals, Long Island, N.Y.), CUDC-101 (Curis, Cambridge,Mass., U.S. Pat. No. 7,547,781), D-69491 (Baxter International,Deerfield, Ill.), Dimercept (Receptor Biologix, Palo Alto, Calif.),EHT-102 (ExonHit Therapeutics, Paris, France), HER2 antagonist (CentgentTherapeutics, San Diego, Calif.), HER/neu vaccine (Corixa, Seattle,Wash.), Herzyme (Sirna Therapeutics, San Francisco, Calif.), HuMax-Her2(Genmab, Copenhagen, Denmark), INSM-18 (Insmed, Richmond, Va.),lapatinib (ditosilatehydrate(N-[3-chloro-4-[(3-fluorobenzyl)oxy]phenyl]-6-[5-[[[2-(methyl-sulfonyl)ethyl]amino]methyl]furan-2-yl]quinazolin-4-aminebis(4-methylbenzenesulfonate)monohydrate) (GlaxoSmithKline, London,England), MP-412 (Mitsubishi Tanabe Pharma Co., Osaka, Japan), mu-4-D-5(Genentech, Oceanside, Calif.), mubritinib(1-[4-[4-[[2-[(E)-2-[4-(trifluoromethyl)phenyl]ethenyl]oxazol-4-yl]methoxy]phenyl]butyl]-1H-1,2,3-triazole)(Takeda Pharmaceuticals, Deerfield, Ill.), neratinib((2E)-N-[4-[[3-chloro-4-[(pyridin-2-yl)methoxy]phenyl]]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide):(Wyeth, Madison, N.J.), pertuzumab (Genentech, Oceanside, Calif.),PX-103.1 (Pharmexa, Copenhagen, Denmark), PX-103.2 (Pharmexa,Copenhagen, Denmark), PX-104.1 (Pharmexa, Copenhagen, Denmark), S-222611(Shionogi, Osaka, Japan), TAK-285 (Takeda Pharmaceuticals, Deerfield,Ill.), trastuzumab (Genentech, Oceanside, Calif.), Trastuzumab-DM1(ImmunoGen, Waltham, Mass.), varlitinib(4-N-[3-chloro-4-(thiazol-2-ylmethoxy)phenyl]-6-N-[(4R)-4-methyl-4,5-dihydrooxazol-2-yl]quinazoline-4,6-diaminebis(4-methylbenzenesulfonate)) (Array BioPharma, Boulder, Colo.), VM-206(ViroMed, Minneapolis, Minn.).

Agents that can affect an activity of HER4 include Dimercept (ReceptorBiologix, Palo Alto, Calif.), neratinib((2E)-N-[4-[[3-chloro-4-[(pyridin-2-yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide)(Wyeth, Madison, N.J.).

Particular non-limiting examples of agents that can bind to and/or alteractivity of other HER family members and can be used in the compositionsand methods provided herein include, without limitation, panitumumab(Amgen, Thousand Oaks, Calif.), erlotinib (Genentech, South SanFrancisco, Calif.; OSI Pharmaceuticals, Long Island, N.Y.; Roche, Basel,Switzerland), lapatinib Glaxo Smith Kline, London, U.K.), pertuzumab(Genentech, South San Francisco, Calif.), trastuzumab (Genentech, SouthSan Francisco, Calif.), cetuximab (ImClone, New York, N.Y.; and BristolMyers Squibb, New York, N.Y.), neratinib (Pfizer Inc., New York, N.Y.),and T-DM1 (Genentech, South San Francisco, Calif.; Roche, Basel,Switzerland), gefitinib (AstraZeneca, London, U.K., and Teva, PetahTikva, Israel). These are described in further detail below.

Panitumumab, marketed as VECTIBIX®, is a fully human monoclonal antibodyspecific to EGF-R. In some embodiments, a combination for treatment ofHER3-associated disease can be U1-1, U1-2, U1-3, U1-4, U1-5, U1-6, U1-7,U1-8, U1-9, U1-10, U1-11, U1-12, U1-13, U1-14, U1-15, U1-16, U1-17,U1-18, U1-19, U1-20, U1-21, U1-22, U1-23, U1-24, U1-25, U1-26, U1-27,U1-28, U1-29, U1-30, U1-31, U1-32, U1-33, U1-34, U1-35, U1-36, U1-37,U1-38, U1-39, U1-40, U1-41, U1-42, U1-43, U1-44, U1-45, U1-46, U1-47,U1-48, U1-49, U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55, U1-57.1,U1-57, U1-58, U1-59, U1-61.1, U1-61, or U1-62, in combination withpanitumumab, or U1-1, U1-2, U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9,U1-10, U1-11, U1-12, U1-13, U1-14, U1-15, U1-16, U1-17, U1-18, U1-19,U1-20, U1-21, U1-22, U1-23, U1-24, U1-25, U1-26, U1-27, U1-28, U1-29,U1-30, U1-31, U1-32, U1-33, U1-34, U1-35, U1-36, U1-37, U1-38, U1-39,U1-40, U1-41, U-42, U1-43, U1-44, U1-45, U1-46, U1-47, U1-48, U1-49,U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55, U1-57.1, U1-57, U1-58,U1-59, U1-61.1, U1-61, or U1-62, U1-1, U1-2, U1-3, U1-4, U1-5, U1-6,U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13, U1-14, U1-15, U1-16,U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23, U1-24, U1-25, U1-26,U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33, U1-34, U1-35, U1-36,U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43, U1-44, U1-45, U1-46,U1-47, U1-48, U1-49, U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55,U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, or U1-62, in combinationwith panitumumab, for treatment of neoplastic disease, such as cancer.Examples of cancer types that may be treated with such combinations arebreast cancer, gastrointestinal cancer, pancreatic cancer, prostatecancer, ovarian cancer, stomach cancer, endometrial cancer, salivarygland cancer, lung cancer, renal cancer, colon cancer, colorectalcancer, thyroid cancer, bladder cancer, glioma, melanoma, metastaticbreast cancer, non-small cell lung cancer, epidermoid carcinoma,fibrosarcoma, melanoma, nasopharyngeal carcinoma, and squamous cellcarcinoma.

Erlotinib (marketed as TARCEVA™) is a drug used to treat NSCLC,pancreatic cancer, and several other types of cancer. Erlotinibspecifically targets the EGF-R tyrosine kinase, binding reversibly tothe ATP binding site of the receptor. In some embodiments, a compositionfor treatment of HER3-associated disease can be U1-1, U1-2, U1-3, U1-4,U1-5, U1-6, U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13, U1-14, U1-15,U1-16, U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23, U1-24, U1-25,U1-26, U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33, U1-34, U1-35,U1-36, U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43, U1-44, U1-45,U1-46, U1-47, U1-48, U1-49, U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55,U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, or U1-62, U1-1, U1-2,U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13,U1-14, U1-15, U1-16, U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23,U1-24, U1-25, U1-26, U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33,U1-34, U1-35, U1-36, U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43,U1-44, U1-45, U1-46, U1-47, U1-48, U1-49, U1-50, U1-51, U1-52, U1-53,U1-55.1, U1-55, U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, orU1-62,U1-49, U1-53 or U1-59, in combination with erlotinib, or U1-1,U1-2, U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9, U1-10, U1-11, U1-12,U1-13, U1-14, U1-15, U1-16, U1-17, U1-18, U1-19, U1-20, U1-21, U1-22,U1-23, U1-24, U1-25, U1-26, U1-27, U1-28, U1-29, U1-30, U1-31, U1-32,U1-33, U1-34, U1-35, U1-36, U1-37, U1-38, U1-39, U1-40, U1-41, U1-42,U1-43, U1-44, U1-45, U1-46, U1-47, U1-48, U1-49, U1-50, U1-51, U1-52,U1-53, U1-55.1, U1-55, U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, orU1-62, U1-1, U1-2, U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9, U1-10,U1-11, U1-12, U1-13, U1-14, U1-15, U1-16, U1-17, U1-18, U1-19, U1-20,U1-21, U1-22, U1-23, U1-24, U1-25, U1-26, U1-27, U1-28, U1-29, U1-30,U1-31, U1-32, U1-33, U1-34, U1-35, U1-36, U1-37, U1-38, U1-39, U1-40,U1-41, U1-42, U1-43, U1-44, U1-45, U1-46, U1-47, U1-48, U1-49, U1-50,U1-51, U1-52, U1-53, U1-55.1, U1-55, U1-57.1, U1-57, U1-58, U1-59,U1-61.1, U1-61, or U1-62,U1-49, U1-53 or U1-59, in combination witherlotinib and other agent(s), for treatment of neoplastic disease, suchas cancer, including but not limited to breast cancer, gastrointestinalcancer, pancreatic cancer, prostate cancer, ovarian cancer, stomachcancer, endometrial cancer, salivary gland cancer, lung cancer, renalcancer, colon cancer, colorectal cancer, thyroid cancer, bladder cancer,glioma, melanoma, metastatic breast cancer, non-small cell lung cancer,epidermoid carcinoma, fibrosarcoma, melanoma, nasopharyngeal carcinoma,or squamous cell carcinoma. In some preferred embodiments, U1-49, U1-53or U1-59 can be used in the treatment of patients with cancers includingnon-small cell lung cancer (NSCLC), locally advanced NSCLC andmetastatic NSCLC after failure of at least one prior chemotherapyregimen, in combination with erlotinib.

Lapatinib (marketed as Tykerb) is an orally active small molecule forthe treatment of solid tumors such as breast cancer. Lapatinib is a dualtyrosine kinase inhibitor that inhibits tyrosine kinase activityassociated with EGF-R and HER2/neu (human EGF-R type 2). In someembodiments, a composition for treatment of HER3-associated disease canbe U1-1, U1-2, U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9, U1-10, U1-11,U1-12, U1-13, U1-14, U1-15, U1-16, U1-17, U1-18, U1-19, U1-20, U1-21,U1-22, U1-23, U1-24, U1-25, U1-26, U1-27, U1-28, U1-29, U1-30, U1-31,U1-32, U1-33, U1-34, U1-35, U1-36, U1-37, U1-38, U1-39, U1-40, U1-41,U1-42, U1-43, U1-44, U1-45, U1-46, U1-47, U1-48, U1-49, U1-50, U1-51,U1-52, U1-53, U1-55.1, U1-55, U1-57.1, U1-57, U1-58, U1-59, U1-61.1,U1-61, or U1-62, U1-1, U1-2, U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9,U1-10, U1-11, U1-12, U1-13, U1-14, U1-15, U1-16, U1-17, U1-18, U1-19,U1-20, U1-21, U1-22, U1-23, U1-24, U1-25, U1-26, U1-27, U1-28, U1-29,U1-30, U1-31, U1-32, U1-33, U1-34, U1-35, U1-36, U1-37, U1-38, U1-39,U1-40, U1-41, U1-42, U1-43, U1-44, U1-45, U1-46, U1-47, U1-48, U1-49,U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55, U1-57.1, U1-57, U1-58,U1-59, U1-61.1, U1-61, or U1-62,U1-49, U1-53 or U1-59, in combinationwith lapatinib, or U1-1, U1-2, U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9,U1-10, U1-11, U1-12, U1-13, U1-14, U1-15, U1-16, U1-17, U1-18, U1-19,U1-20, U1-21, U1-22, U1-23, U1-24, U1-25, U1-26, U1-27, U1-28, U1-29,U1-30, U1-31, U1-32, U1-33, U1-34, U1-35, U1-36, U1-37, U1-38, U1-39,U1-40, U1-41, U1-42, U1-43, U1-44, U1-45, U1-46, U1-47, U1-48, U1-49,U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55, U1-57.1, U1-57, U1-58,U1-59, U1-61.1, U1-61, or U1-62, U1-1, U1-2, U1-3, U1-4, U1-5, U1-6,U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13, U1-14, U1-15, U1-16,U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23, U1-24, U1-25, U1-26,U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33, U1-34, U1-35, U1-36,U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43, U1-44, U1-45, U1-46,U1-47, U1-48, U1-49, U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55,U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, or U1-62,U1-49, U1-53 orU1-59, in combination with lapatinib and other agent(s) such ascapecitabine, for treatment of neoplastic disease, such as cancer,wherein the cancer is, for example, breast cancer, gastrointestinalcancer, pancreatic cancer, prostate cancer, ovarian cancer, stomachcancer, endometrial cancer, salivary gland cancer, lung cancer, renalcancer, colon cancer, colorectal cancer, thyroid cancer, bladder cancer,glioma, melanoma, metastatic breast cancer, non-small cell lung cancer,epidermoid carcinoma, fibrosarcoma, melanoma, nasopharyngeal carcinoma,or squamous cell carcinoma. In some preferred embodiments, U1-49, U1-53or U1-59 can be used in the treatment of patients with cancers includingbreast cancer and metastatic breast cancer whose tumors express oroverexpress the HER-2 protein and who have received prior chemotherapyincluding an anthracycline (for example, doxorubicin or related agent)and/or a taxane (for example, paclitaxel or docetaxel), and trastuzumab,in combination with lapatinib, or, in combination with lapatinib andcapecitabine.

Trastuzumab (also known as HERCEPTIN®) is a humanized monoclonalantibody that interferes with the HER2/neu receptor. In someembodiments, a composition for treatment of HER3-associated disease canbe U1-1, U1-2, U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9, U1-10, U1-11,U1-12, U1-13, U1-14, U1-15, U1-16, U1-17, U1-18, U1-19, U1-20, U1-21,U1-22, U1-23, U1-24, U1-25, U1-26, U1-27, U1-28, U1-29, U1-30, U1-31,U1-32, U1-33, U1-34, U1-35, U1-36, U1-37, U1-38, U1-39, U1-40, U1-41,U1-42, U1-43, U1-44, U1-45, U1-46, U1-47, U1-48, U1-49, U1-50, U1-51,U1-52, U1-53, U1-55.1, U1-55, U1-57.1, U1-57, U1-58, U1-59, U1-61.1,U1-61, or U1-62, U1-1, U1-2, U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9,U1-10, U1-11, U1-12, U1-13, U1-14, U1-15, U1-16, U1-17, U1-18, U1-19,U1-20, U1-21, U1-22, U1-23, U1-24, U1-25, U1-26, U1-27, U1-28, U1-29,U1-30, U1-31, U1-32, U1-33, U1-34, U1-35, U1-36, U1-37, U1-38, U1-39,U1-40, U1-41, U1-42, U1-43, U1-44, U1-45, U1-46, U1-47, U1-48, U1-49,U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55, U1-57.1, U1-57, U1-58,U1-59, U1-61.1, U1-61, or U1-62,U1-49, U1-53 or U1-59, in combinationwith trastuzumab, or U1-1, U1-2, U1-3, U1-4, U1-5, U1-6, U1-7, U1-8,U1-9, U1-10, U1-11, U1-12, U1-13, U1-14, U1-15, U1-16, U1-17, U1-18,U1-19, U1-20, U1-21, U1-22, U1-23, U1-24, U1-25, U1-26, U1-27, U1-28,U1-29, U1-30, U1-31, U1-32, U1-33, U1-34, U1-35, U1-36, U1-37, U1-38,U1-39, U1-40, U1-41, U1-42, U1-43, U1-44, U1-45, U1-46, U1-47, U1-48,U1-49, U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55, U1-57.1, U1-57,U1-58, U1-59, U1-61.1, U1-61, or U1-62, U1-1, U1-2, U1-3, U1-4, U1-5,U1-6, U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13, U1-14, U1-15, U1-16,U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23, U1-24, U1-25, U1-26,U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33, U1-34, U1-35, U1-36,U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43, U1-44, U1-45, U1-46,U1-47, U1-48, U1-49, U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55,U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, or U1-62,U1-49, U1-53 orU1-59, in combination with trastuzumab and other agent(s) such asdocetaxel or paclitaxel, for treatment of neoplastic disease, such ascancer, such as breast cancer, gastrointestinal cancer, pancreaticcancer, prostate cancer, ovarian cancer, stomach cancer, endometrialcancer, salivary gland cancer, lung cancer, renal cancer, colon cancer,colorectal cancer, thyroid cancer, bladder cancer, glioma, melanoma,metastatic breast cancer, non-small cell lung cancer, epidermoidcarcinoma, fibrosarcoma, melanoma, nasopharyngeal carcinoma, or squamouscell carcinoma. In some preferred embodiments, U1-49, U1-53 or U1-59 canbe used in the treatment of patients with cancers including breastcancer and metastatic breast cancer whose tumors express or overexpressthe HER-2 protein and who have not received chemotherapy for their(metastatic) disease, in combination with trastuzumab and paclitaxel,or, in combination with trastuzumab and docetaxel.

T-DM1 is an antibody-drug conjugate that includes trastuzumab chemicallylinked to a potent antimicrotubule drug (DM1) derived from maytansine.Maytansine has been used as a free drug, and has shown effectiveness in,e.g., breast and lung cancer patients. The non-reducible thioether MCClinker is used in T-DM1, providing a stable bond between trastuzumab andDM1, prolonging exposure, and reducing the toxicity of T-DM1 whilemaintaining activity. In some embodiments, a method for treatment ofHER3-associated disease can include administering U1-1, U1-2, U1-3,U1-4, U1-5, U1-6, U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13, U1-14,U1-15, U1-16, U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23, U1-24,U1-25, U1-26, U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33, U1-34,U1-35, U1-36, U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43, U1-44,U1-45, U1-46, U1-47, U1-48, U1-49, U1-50, U1-51, U1-52, U1-53, U1-55.1,U1-55, U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, or U1-62, U1-1,U1-2, U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9, U1-10, U1-11, U1-12,U1-13, U1-14, U1-15, U1-16, U1-17, U1-18, U1-19, U1-20, U1-21, U1-22,U1-23, U1-24, U1-25, U1-26, U1-27, U1-28, U1-29, U1-30, U1-31, U1-32,U1-33, U1-34, U1-35, U1-36, U1-37, U1-38, U1-39, U1-40, U1-41, U1-42,U1-43, U1-44, U1-45, U1-46, U1-47, U1-48, U1-49, U1-50, U1-51, U1-52,U1-53, U1-55.1, U1-55, U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, orU1-62, U1-49, U1-53 or U1-59, in combination with T-DM1 (e.g., eithersimultaneously or separately), or U1-1, U1-2, U1-3, U1-4, U1-5, U1-6,U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13, U1-14, U1-15, U1-16,U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23, U1-24, U1-25, U1-26,U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33, U1-34, U1-35, U1-36,U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43, U1-44, U1-45, U1-46,U1-47, U1-48, U1-49, U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55,U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, or U1-62, U1-1, U1-2,U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13,U1-14, U1-15, U1-16, U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23,U1-24, U1-25, U1-26, U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33,U1-34, U1-35, U1-36, U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43,U1-44, U1-45, U1-46, U1-47, U1-48, U1-49, U1-50, U1-51,U1-52, U1-53,U1-55.1, U1-55, U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, orU1-62,U1-49, U 1-53 or U 1-59, in combination with T-DM1 and otheragent(s) such as docetaxel or paclitaxel, for treatment of neoplasticdisease, such as cancer, including cancers such as gastrointestinalcancer, pancreatic cancer, prostate cancer, ovarian cancer, stomachcancer, endometrial cancer, salivary gland cancer, kidney cancer, coloncancer, thyroid cancer, bladder cancer, glioma, melanoma, lung cancerincluding non-small cell lung cancer, colorectal cancer, and/or breastcancer including metastatic breast cancer. In some preferredembodiments, U1-49, U1-53 or U1-59 can be used in the treatment ofpatients with cancers including breast cancer and metastatic breastcancer whose tumors express or overexpress the HER-2 protein and whohave not received chemotherapy for their (metastatic) disease, incombination with T-DM1 and paclitaxel, or, in combination with T-DM1 anddocetaxel.

Pertuzumab (2C4) (marketed or to be marketed as OMNITARG™) is amonoclonal antibody that inhibits the dimerization of HER2 with otherHER receptors. In some embodiments, a composition for treatment ofHER3-associated disease can be U1-1, U1-2, U1-3, U1-4, U1-5, U1-6, U1-7,U1-8, U1-9, U1-10, U1-11, U1-12, U1-13, U1-14, U1-15, U1-16, U1-17,U1-18, U1-19, U1-20, U1-21, U1-22, U1-23, U1-24, U1-25, U1-26, U1-27,U1-28, U1-29, U1-30, U1-31, U1-32, U1-33, U1-34, U1-35, U1-36, U1-37,U1-38, U1-39, U1-40, U1-41, U1-42, U1-43, U1-44, U1-45, U1-46, U1-47,U1-48, U1-49, U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55, U1-57.1,U1-57, U1-58, U1-59, U1-61.1, U1-61, or U1-62, U1-1, U1-2, U1-3, U1-4,U1-5, U1-6, U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13, U1-14, U1-15,U1-16, U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23, U1-24, U1-25,U1-26, U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33, U1-34, U1-35,U1-36, U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43, U1-44, U1-45,U1-46, U1-47, U1-48, U1-49, U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55,U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, or U1-62,U1-49, U1-53 orU1-59, in combination with pertuzumab, or U1-1, U1-2, U1-3, U1-4, U1-5,U1-6, U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13, U1-14, U1-15, U1-16,U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23, U1-24, U1-25, U1-26,U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33, U1-34, U1-35, U1-36,U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43, U1-44, U1-45, U1-46,U1-47, U1-48, U1-49, U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55,U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, or U1-62, U1-1, U1-2,U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13,U1-14, U1-15, U1-16, U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23,U1-24, U1-25, U1-26, U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33,U1-34, U1-35, U1-36, U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43,U1-44, U1-45, U1-46, U1-47, U1-48, U1-49, U1-50, U1-51, U1-52, U1-53,U1-55.1, U1-55, U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, or U1-62,U1-49, U1-53 or U1-59, in combination with pertuzumab and otheragent(s), for treatment of neoplastic disease, such as cancer,including, e.g., breast cancer, gastrointestinal cancer, pancreaticcancer, prostate cancer, ovarian cancer, stomach cancer, endometrialcancer, salivary gland cancer, lung cancer, renal cancer, colon cancer,colorectal cancer, thyroid cancer, bladder cancer, glioma, melanoma,metastatic breast cancer, non-small cell lung cancer, epidermoidcarcinoma, fibrosarcoma, melanoma, nasopharyngeal carcinoma, andsquamous cell carcinoma.

Cetuximab (marketed as ERBITUX®) is a chimeric (mouse/human) monoclonalantibody that binds to and inhibits EGF-R. In some embodiments, acomposition for treatment of HER3-associated disease can be U1-1, U1-2,U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13,U1-14, U1-15, U1-16, U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23,U1-24, U1-25, U1-26, U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33,U1-34, U1-35, U1-36, U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43,U1-44, U1-45, U1-46, U1-47, U1-48, U1-49, U1-50, U1-51, U1-52, U1-53,U1-55.1, U1-55, U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, or U1-62,U1-1, U1-2, U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9, U1-10, U1-11,U1-12, U1-13, U1-14, U1-15, U1-16, U1-17, U1-18, U1-19, U1-20, U1-21,U1-22, U1-23, U1-24, U1-25, U1-26, U1-27, U1-28, U1-29, U1-30, U1-31,U1-32, U1-33, U1-34, U1-35, U1-36, U1-37, U1-38, U1-39, U1-40, U1-41,U1-42, U1-43, U1-44, U1-45, U1-46, U1-47, U1-48, U1-49, U1-50, U1-51,U1-52, U1-53, U1-55.1, U1-55, U1-57.1, U1-57, U1-58, U1-59, U1-61.1,U1-61, or U1-62,U1-49, U1-53 or U1-59, in combination with cetuximab, orU1-1, U1-2, U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9, U1-10, U1-11,U1-12, U1-13, U1-14, U1-15, U1-16, U1-17, U1-18, U1-19, U1-20, U1-21,U1-22, U1-23, U1-24, U1-25, U1-26, U1-27, U1-28, U1-29, U1-30, U1-31,U1-32, U1-33, U1-34, U1-35, U1-36, U1-37, U1-38, U1-39, U1-40, U1-41,U1-42, U1-43, U1-44, U1-45, U1-46, U1-47, U1-48, U1-49, U1-50, U1-51,U1-52, U1-53, U1-55.1, U1-55, U1-57.1, U1-57, U1-58, U1-59, U1-61.1,U1-61, or U1-62, U1-1, U1-2, U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9,U1-10, U1-11, U1-12, U1-13, U1-14, U1-15, U1-16, U1-17, U1-18, U1-19,U1-20, U1-21, U1-22, U1-23, U1-24, U1-25, U1-26, U1-27, U1-28, U1-29,U1-30, U1-31, U1-32, U1-33, U1-34, U1-35, U1-36, U1-37, U1-38, U1-39,U1-40, U1-41, U1-42, U1-43, U1-44, U1-45, U1-46, U1-47, U1-48, U1-49,U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55, U1- 57.1, U1-57, U1-58,U1-59, U1-61.1, U1-61, or U1-62,U1-49, U1-53 or U1-59, in combinationwith cetuximab for treatment of neoplastic disease, such as cancer,including, e.g., breast cancer, gastrointestinal cancer, pancreaticcancer, prostate cancer, ovarian cancer, stomach cancer, endometrialcancer, salivary gland cancer, lung cancer, renal cancer, colon cancer,colorectal cancer, thyroid cancer, bladder cancer, glioma, melanoma,metastatic breast cancer, non-small cell lung cancer, epidermoidcarcinoma, fibrosarcoma, melanoma, nasopharyngeal carcinoma, andsquamous cell carcinoma. In some preferred embodiments, U1-49, U1-53 orU1-59 can be used in the treatment of patients with cancers includingcolorectal cancer and metastatic colorectal cancer after failure of5-fluorouracil-based chemotherapy, in combination with cetuximab andirinotecan.

Gefitinib (marketed as IRESSA®) is a drug that acts in a similar mannerto erlotinib. Gefitinib selectively inhibits EGF-R's tyrosine kinasedomain. In some embodiments, a composition for treatment ofHER3-associated disease can be U1-1, U1-2, U1-3, U1-4, U1-5, U1-6, U1-7,U1-8, U1-9, U1-10, U1-11, U1-12, U1-13, U1-14, U1-15, U1-16, U1-17,U1-18, U1-19, U1-20, U1-21, U1-22, U1-23, U1-24, U1-25, U1-26, U1-27,U1-28, U1-29, U1-30, U1-31, U1-32, U1-33, U1-34, U1-35, U1-36, U1-37,U1-38, U1-39, U1-40, U1-41, U1-42, U1-43, U1-44, U1-45, U1-46, U1-47,U1-48, U1-49, U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55, U1-57.1,U1-57, U1-58, U1-59, U1-61.1, U1-61, or U1-62, U1-1, U1-2, U1-3, U1-4,U1-5, U1-6, U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13, U1-14, U1-15,U1-16, U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23, U1-24, U1-25,U1-26, U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33, U1-34, U1-35,U1-36, U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43, U1-44, U1-45,U1-46, U1-47, U1-48, U1-49, U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55,U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, or U1-62,U1-49, U1-53 orU1-59, in combination with gefitinib, or U1-1, U1-2, U1-3, U1-4, U1-5,U1-6, U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13, U1-14, U1-15, U1-16,U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23, U1-24, U1-25, U1-26,U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33, U1-34, U1-35, U1-36,U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43, U1-44, U1-45, U1-46,U1-47, U1-48, U1-49, U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55,U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, or U1-62, U1-1, U1-2,U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13,U1-14, U1-15, U1-16, U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23,U1-24, U1-25, U1-26, U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33,U1-34, U1-35, U1-36, U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43,U1-44, U1-45, U1-46, U1-47, U1-48, U1-49, U1-50, U1-51, U1-52, U1-53,U1-55.1, U1-55, U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, orU1-62,U1-49, U1-53 or U1-59, in combination with gefitinib and otheragent(s), for treatment of neoplastic disease such as cancer, including,e.g., breast cancer, gastrointestinal cancer, pancreatic cancer,prostate cancer, ovarian cancer, stomach cancer, endometrial cancer,salivary gland cancer, lung cancer, renal cancer, colon cancer,colorectal cancer, thyroid cancer, bladder cancer, glioma, melanoma,metastatic breast cancer, non-small cell lung cancer, epidermoidcarcinoma, fibrosarcoma, melanoma, nasopharyngeal carcinoma, andsquamous cell carcinoma.

Neratinib is an inhibitor of the HER-2 receptor tyrosine kinase.Neratinib binds irreversibly to the HER-2 receptor and thereby reducesautophosphorylation in cells, apparently by targeting a cysteine residuein the ATP-binding pocket of the receptor. Treatment of cells withneratinib results in inhibition of downstream signal transduction eventsand cell cycle regulatory pathways, arrest at the G1-S-phase transitionof the cell cycle, and ultimately decreased cellular proliferation. Inaddition, neratinib inhibits the EGF-R kinase and proliferation ofEGF-R-dependent cells. In some embodiments, a method for treatment ofHER3-associated disease can include administration of U1-1, U1-2, U1-3,U1-4, U1-5, U1-6, U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13, U1-14,U1-15, U1-16, U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23, U1-24,U1-25, U1-26, U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33, U1-34,U1-35, U1-36, U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43, U1-44,U1-45, U1-46, U1-47, U1-48, U1-49, U1-50, U1-51, U1-52, U1-53, U1-55.1,U1-55, U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, or U1-62, U1-1,U1-2, U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9, U1-10, U1-11, U1-12,U1-13, U1-14, U1-15, U1-16, U1-17, U1-18, U1-19, U1-20, U1-21, U1-22,U1-23, U1-24, U1-25, U1-26, U1-27, U1-28, U1-29, U1-30, U1-31, U1-32,U1-33, U1-34, U1-35, U1-36, U1-37, U1-38, U1-39, U1-40, U1-41, U1-42,U1-43, U1-44, U1-45, U1-46, U1-47, U1-48, U1-49, U1-50, U1-51, U1-52,U1-53, U1-55.1, U1-55, U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, orU1-62,U1-49, U1-53 or U1-59, in combination with neratinib (e.g.,simultaneously or separately), or U1-1, U1-2, U1-3, U1-4, U1-5, U1-6,U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13, U1-14, U1-15, U1-16,U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23, U1-24, U1-25, U1-26,U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33, U1-34, U1-35, U1-36,U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43, U1-44, U1-45, U1-46,U1-47, U1-48, U1-49, U1-50, U1-51, U1-52, U1-53, U1-55.1, U1-55,U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, or U1-62, U1-1, U1-2,U1-3, U1-4, U1-5, U1-6, U1-7, U1-8, U1-9, U1-10, U1-11, U1-12, U1-13,U1-14, U1-15, U1-16, U1-17, U1-18, U1-19, U1-20, U1-21, U1-22, U1-23,U1-24, U1-25, U1-26, U1-27, U1-28, U1-29, U1-30, U1-31, U1-32, U1-33,U1-34, U1-35, U1-36, U1-37, U1-38, U1-39, U1-40, U1-41, U1-42, U1-43,U1-44, U1-45, U1-46, U1-47, U1-48, U1-49, U1-50, U1-51, U1-52, U1-53,U1-55.1, U1-55, U1-57.1, U1-57, U1-58, U1-59, U1-61.1, U1-61, orU1-62,U1-49, U1-53 or U1-59, in combination with neratinib and otheragent(s), for treatment of neoplastic disease such as cancer, including,e.g., gastrointestinal cancer, pancreatic cancer, prostate cancer,ovarian cancer, stomach cancer, endometrial cancer, salivary glandcancer, kidney cancer, colon cancer, thyroid cancer, bladder cancer,glioma, melanoma, lung cancer including non-small cell lung cancer,colorectal cancer and/or breast cancer including metastatic breastcancer.

4. Additional Agents to be used in the Compositions and MethodsDisclosed Herein

Additional agents may be added to the first and second agent binding toHER-3, and binding to and/or inhibiting another member of the HERfamily, respectively, as disclosed herein. These, in some embodiments,will be chemotherapeutic drugs. The additional agents to be used in thecompositions and methods disclosed herein can be also used as the secondagent(s) in place of that binding to and/or inhibiting another member ofthe HER family in the present inventions. In other words, the firstagent binding to HER3 can be used in certain treatment in combinationwith any of the additional agents described hereinafter without/insteadfor the second agent binding to and/or inhibiting another HER family.

For example, agents that act as microtubule stimulants include NK-105(paclitaxel)[(−)-(1S,2R,3S,4S,5R,7S,8S,10R,13S)-4,10-diacetoxy-2-benzoyloxy-5,20-epoxy-1,7-dihydroxy-9-oxotax-11-en-13-yl(2R,3S)-3-benzoylamino-2-hydroxy-3-phenylpropionate] (NanoCarrier,Chiba, Japan), milataxel(1,10β-dihydroxy-9-oxo-5β,20-epoxy-3zeta-tax-11-ene-2α,4,7β13α-tetrayl4-acetate 2-benzoate13-[(2R,3R)-3-(tert-butoxycarbonylamino)-3-(furan-2-yl)-2-hydroxypropanoate]7-propanoate) (Taxolog, Fairfield, N.J.), laulimalide (KosanBiosciences, Hayward, Calif. (B-M Squibb)), sarcodictyin A(3-(1-methylimidazol-4-yl)-2(E)-propenoic acid(1R,4aR,6S,7S,10R,12aR)-11-methoxycarbonyl-7,10-epoxy-10-hydroxy-1-isopropyl-4,7-dimethyl-1,2,4a,5,6,7,10,12a-octahydrobenzocyclododecen-6-ylester) (Pfizer, New York, N.Y.), simotaxel((2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-7,11-methano-1H-cyclodeca[3,4]benz[1,2-b]oxete-6,9,12,12b-tetrayl12b-acetate 12-benzoate 6-cyclopentanecarboxylate9-[(2R,3R)-2-hydroxy-3-[[(1-methylethoxy)carbonyl]amino]-3-(thiophen-2-yl)propanoate])(Taxolog, Fairfield, N.J.), SYN-2001 (CLL Pharma, Nice, France), TL-310(Taxolog, Fairfield, N.J.), TL1836 (Taxolog, Fairfield, N.J.), tesetaxel(2′-[(dimethylamino)methyl]-1-hydroxy-5β,20-epoxy-9α,10α-dihydro[1,3]dioxolo[4′,5′:9,10]tax-11-ene-2α,4,13α-triyl4-acetate2-benzoate13-[(2R,3S)-3-[(tert-butoxycarbonyl)amino]-3-(3-fluoropyridin-2-yl)-2-hydroxypropanoate)(Daiichi Sankyo, Tokyo, Japan), TL-1892 (Taxolog, Fairfield, N.J.),TPI-287 ((2′R,3′S)-2′-hydroxy-N-carboxy-3′-amino-5′-methyl-hexanoic,N-tert-butyl ester, 13 ester5β-20-epoxy-1,2α,4,7β,9α,10α,13α-heptahydroxy-4,10-diacetate-2-benzoate-7,9-acroleinacetal-tax-11-ene (Tapestry Pharmaceuticals, Boulder, Colo.), ortataxel(2aR-[2aα,4β,4aβ,6β,9α(2R,3S),10β,11β,12α,12aα,12bα]-3-(tert-butoxycarbonylamino)-2-hydroxy-5-methyl-hexanoicacid6,12b-diacetoxy-12-benzoyloxy-10,11-carbonyldioxy-4-hydroxy-4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-1H-7,11-methanocyclodeca[3,4]benz[1,2-b]oxet-9-ylester) (Indena, Milan, Italy), paclitaxel poliglumex(L-pyroglutamylpoly-L-glutamyl-L-glutamic acid partially γ-esterifiedwith(1R,2S)-2-(benzoylamino)-1-[[[(2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-6,12b-bis(acetyloxy)-12-(benzoyloxy)-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-7,11-methano-1H-cyclodeca[3,4]benzo[1,2-b]oxet-9-yl]oxy]carbonyl]-2-phenylethyl)(Cell Therapeutics, Seattle, Wash.), paclitaxel protein-bound particles(paclitaxel:(−)-(1S,2R,3S,4S,5R,7S,8S,10R,13S)-4,10-diacetoxy-2-benzoyloxy-5,20-epoxy-1,7-dihydroxy-9-oxotax-11-en-13-yl(2R,3S)-3-benzoylamino-2-hydroxy-3-phenylpropionate) (AbraxisBioScience, Los Angeles, Calif.), paclitaxel (NCI)((−)-(1S,2R,3S,4S,5R,7S,8S,10R,13S)-4,10-diacetoxy-2-benzoyloxy-5,20-epoxy-1,7-dihydroxy-9-oxotax-11-en-13-yl(2R,3S)-3-benzoylamino-2-hydroxy-3-phenylpropionate) (NCI (NIH)),paclitaxel (NeoPharm, Lake Bluff, Ill.)((−)-(1S,2R,3S,4S,5R,7S,8S,10R,13S)-4,10-diacetoxy-2-benzoyloxy-5,20-epoxy-1,7-dihydroxy-9-oxotax-11-en-13-yl(2R,3S)-3-benzoylamino-2-hydroxy-3-phenylpropionate) (NeoPharm, LakeBluff, Ill.), patupilone((1S,3S,7S,10R,11S,12S,16R)-7,11-dihydroxy-8,8,10,12,16-pentamethyl-3-[(1E)-1-(2-methyl-1,3-thiazol-4-yl)prop-1-en-2-yl]-4,17-dioxabicyclo[14.1.0]heptadecane-5,9-dione) (US Publication No. 2003/0104625,Novartis, Basel, Switzerland), PEG-paclitaxel (Enzo Pharmaceuticals,Long Island, N.Y.), docetaxel hydrate((−)-(1S,2S,3R,4S,5R,7S,8S,10R,13S)-4-acetoxy-2-benzoyloxy-5,20-epoxy-1,7,10-trihydroxy-9-oxotax-11-ene-13-yl(2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-3-phenylpropionatetrihydrate) (Sanofi-Aventis, Bridgewater, N.J.), eleutherobin(3-(1-methylimidazol-4-yl)-2(E)-propenoic acid(1R,4aR,6S,7S,10R,12aR)-11-(2-O-acetyl-β-D-arabinopyranosyloxymethyl)-7,10-epoxy-1-isopropyl-10-methoxy-4,7-dimethyl-1,2,4a,5,6,7,10,12a-octahydrobenzocyclododecen-6-ylester) (Bristol-Myers Squibb, New York, N.Y.), IDN-5390 (Indena, Milan,Italy), ixabepilone((1S,3S,7S,10R,11S,12S,16R)-7,11-dihydroxy-8,8,10,12,16-pentamethyl-3-[(1E)-1-methyl-2-(2-methylthiazol-4-yl)ethenyl]-17-oxa-4-azabicyclo[14.1.0]heptadecane-5,9-dione)(Bristol-Myers Squibb, New York, N.Y.), KOS-1584 (Kosan Biosciences,Hayward, Calif. (B-M Squibb)), KOS-1803 (17-iso-oxazole26-trifluoro-9,10-dehydro-12,13-desoxy-epothilone B) (Kosan Biosciences,Hayward, Calif. (B-M Squibb)), KOS-862 (Kosan Biosciences, Hayward,Calif. (B-M Squibb); U.S. Pat. Nos. 6,204,388 and 6,303,342), larotaxel(1-hydroxy-9-oxo-5β,20-epoxy-7β,19-cyclotax-11-ene-2α,4,10β,13α-tetrayl4,10-diacetate 2-benzoate13-[(2R,3S)-3-[(tert-butoxycarbonyl)amino]-2-hydroxy-3-phenylpropanoate]dehydrate)(Sanofi-Aventis, Bridgewater, N.J., PCT Publication Nos. WO 95/26961 andWO 96/1259), ANG-1005 (Angiopep-2/paclitaxel conjugate) (AngioChem,Montreal, Canada, U.S. Pat. No. 7,557,182), BMS-184476 (Bristol-MyersSquibb, New York, N.Y., EP Publication No. 639577), BMS-188797(Bristol-Myers Squibb, New York, N.Y.), BMS-275183(3′-tert-butyl-3′-N-tert-butyloxycarbonyl-4-deacetyl-3′-dephenyl-3′-N-debenzoyl-4-O-methyoxy-carbonyl-paclitaxel)(Bristol-Myers Squibb, New York, N.Y.), BMS-310705 (Bristol-MyersSquibb, New York, N.Y.), BMS-753493 (Bristol-Myers Squibb, New York,N.Y.), cabazitaxel(1-hydroxy-7β,10β-dimethoxy-9-oxo-5β,20-epoxytax-11-ene-2α,4,13α-triyl4-acetate 2-benzoate13-[(2R,3S)-3-[[(tertbutoxy)carbonyl]amino]-2-hydroxy-3-phenylpropanoate])(Sanofi-Aventis, Bridgewater, N.J.), DHA-paclitaxel (Protarga, King ofPrussia, Pa., TAXOPREXIN®), disermolide([3S-[3α,4β,5β,6α(2R*,3Z,5R*,6R*,7S*,8Z,11R*,12S*,13S*,14S*,15R*,16E)]]-6-[14[(aminocarbonyl)oxy]-2,6,12-trihydroxy-5,7,9,11,13,15-hexamethyl-3,8,16,18-nonadecatetraenyl]tetrahydro-4-hydroxy-3,5-dimethyl-2H-pyran-2-one)(Novartis, Basel, Switzerland, U.S. Pat. Nos. 4,939,168 and 5,681,847).Some of these microtubule stimulants have a taxane ring in theirchemical structures; such compounds having a taxane ring are referred as“taxanes” herein.

Anthracyclins include actinomycins such as actinomycin D (Dactinomycin:2-amino-N,N′-bis[(6S,9R,10S,13R,18aS)-6,13-diisopropyl-2,5,9-trimethyl-1,4,7,11,14-pentaoxohexadecahydro-1H-pyrrolo[2,1-i][1,4,7,10,13]oxatetraazacyclohexadecin-10-yl]-4,6-dimethyl-3-oxo-3H-phenoxazine-1,9-dicarboxamide),bleomycin (bleomycin hydrochloride:(3-{[(2′-{(5S,8S,9S,10R,13S)-15-{6-amino-2-[(1S)-3-amino-1-{[(2S)-2,3-diamino-3-oxopropyl]amino}-3-oxopropyl]-5-methylpyrimidin-4-yl}-13-[{[(2R,3S,4S,5S,6S)-3-{[(2R,3S,4S,5R,6R)-4-(carbamoyloxy)-3,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl]oxy}-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl]oxy}(1H-imidazol-5-yl)methyl]-9-hydroxy-5-[(1R)-1-hydroxyethyl]-8,10-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazapentadec-1-yl}-2,4′-bi-1,3-thiazol-4-yl)carbonyl]amino}propyl)(dimethyl)sulfonium),daunorubicin hydrochloride (daunorubicin:8S-cis)-8-Acetyl-10-((3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)oxy)-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacenedionehydrochloride), doxorubicin hydrochloride (doxorubicin:(8S,10S)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-8-glycoloyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacenedionehydrochloride) (Alza, Mountain View, Calif.), idarubicin hydrochloride((7S,9S)-9-acetyl-7,8,9,10-tetrahydro-6,7,9,11-tetrahydroxy-7-O-(2,3,6-trideoxy-3-amino-α-L-lyxo-hexopyranosyl)-5,12-naphthacenedionehydrochloride) (Pfizer, New York, N.Y., U.S. Pat. Nos. 4,046,878 and4,471,052), and mitomycin((1aS,8S,8aR,8bR)-6-Amino-4,7-dioxo-1,1a,2,8,8a,8b-hexahydro-8a-methoxy-5-methylazirino[2,3:3,4]pyrrolo[1,2-α]indol-8-ylmethylcarbamate)(Kyowa-Hakko-Kirin, Tokyo, Japan).

Cisplatin and gemcitabine are chemotherapeutic agents. Cisplatin orcis-diamminedichloroplatinum(II) is a platinum-based drug used to treatvarious types of cancers. The cisplatin platinum complex reacts in vivo,binding to and causing crosslinking of DNA, which ultimately triggersapoptosis. Gemcitabine is a nucleoside analog in which the hydrogenatoms on the 2′ carbons of deoxycytidine are replaced by fluorine atoms.Like fluorouracil and other pyrimidine analogues, gemcitabine replacescytidine during DNA replication, which arrests tumor growth sincefurther nucleosides cannot be attached to the “faulty” nucleoside,resulting in apoptosis. Gemcitabine is marketed as GEMZAR® by Eli Lillyand Company (Indianapolis, Ind.). In some embodiments, a combination fortreatment of HER3-associated disease can be: U1-49, U1-53 or U1-59 incombination with a second agent as described herein and cisplatin orgemcitabine and other agent(s), for treatment of cancer which isgastrointestinal cancer, pancreatic cancer, prostate cancer, ovariancancer, stomach cancer, endometrial cancer, salivary gland cancer,kidney cancer, colon cancer, thyroid cancer, bladder cancer, glioma,melanoma, lung cancer including non-small cell lung cancer, colorectalcancer and/or breast cancer including metastatic breast cancer.

Capecitabine(pentyl[1-(3,4-dihydroxy-5-methyl-tetrahydrofuran-2-yl)-5-fluoro-2-oxo-1H-pyrimidin-4-yl]aminomethanoate,Xeloda, Roche) is an orally-administered chemotherapeutic agent.Capecitabine is a prodrug that is enzymatically converted to5-fluorouracil in the tumor, where it inhibits DNA synthesis and slowsgrowth of tumor tissue. In some embodiments, a combination for treatmentof HER3-associated disease can be: U1-49, U1-53 or U1-59 in combinationwith a second agent as described herein (e.g., lapatanib) andcapecitabine for treatment of cancer, wherein the cancer isgastrointestinal cancer, pancreatic cancer, prostate cancer, ovariancancer, stomach cancer, endometrial cancer, salivary gland cancer,kidney cancer, colon cancer, thyroid cancer, bladder cancer, glioma,melanoma, lung cancer including non-small cell lung cancer, colorectalcancer and/or breast cancer including metastatic breast cancer. In somecases, such a combination can be administered after failure of priortreatment with an anthracyclin or taxane, for example. In some preferredembodiments, U1-49, U1-53 or U1-59 can be used in the treatment ofpatients with cancers including breast cancer and metastatic breastcancer whose tumors express or overexpress the HER-2 protein and whohave received prior chemotherapy including an anthracycline (forexample, doxorubicin or related agent), and/or a taxane (for example,paclitaxel or docetaxel), and trastuzumab, in combination with lapatiniband capecitabine.

Docetaxel ((2R,3S)-N-carboxy-3-phenylisoserine, N-tert-butyl ester,13-ester with 5,20-epoxy-1,2,4,7,10,13-hexahydroxytax-11-en-9-one4-acetate 2-benzoate, trihydrate) and paclitaxel((2α,4α,5β,7β,10β,13α)-4,10-bis(acetyloxy)-13-{[(2R,3S)-3-(benzoylamino)-2-hydroxy-3-phenylpropanoyl]oxy}-1,7-dihydroxy-9-oxo-5,20-epoxytax-11-en-2-ylbe) are chemotherapeutic agents. Docetaxel is marketed as Taxotere bySanofi Aventis. Paclitaxel is marketed as Taxol by Bristol-Myers Squibb.In the formulation of Taxol, paclitaxel is dissolved in Cremophor EL andethanol, as a delivery agent. A formulation in which paclitaxel is boundto albumin is marketed as Abraxane. In some embodiments, a combinationfor treatment of HER3-associated disease can be: U1-49, U1-53 or U1-59in combination with a second agent as described herein (e.g.,trastuzumab) and docetaxel or paclitaxel and other agent(s) such astrastuzumab, for treatment of cancer, wherein the cancer isgastrointestinal cancer, pancreatic cancer, prostate cancer, ovariancancer, stomach cancer, endometrial cancer, salivary gland cancer,kidney cancer, colon cancer, thyroid cancer, bladder cancer, glioma,melanoma, lung cancer including non-small cell lung cancer, colorectalcancer and/or breast cancer including metastatic breast cancer. In somepreferred embodiments, U1-49, U1-53 or U1-59 can be use in the treatmentof patients with cancers including breast cancer and metastatic breastcancer whose tumors express or overexpress the HER-2 protein and whohave not received chemotherapy for their (metastatic) disease, incombination with trastuzumab and paclitaxel, in combination with T-DM1and paclitaxel, in combination with trastuzumab and docetaxel, or, incombination with T-DM1 and docetaxel.

Doxorubicin hydrochloride liposome injection is marketed as Doxil, aliposome formulation comprising doxorubicin chloride. In someembodiments, a combination treatment for HER-3-associated disease caninclude administering U1-49, U1-53 or U1-59 in combination with a secondagent as described herein and doxorubicin hydrochloride liposomeinjection, with or without one or more other agents such as paclitaxelor platinum-based chemotherapeutic agents, for treatment of cancer suchas breast cancer, gastrointestinal cancer, pancreatic cancer, prostatecancer, ovarian cancer, stomach cancer, endometrial cancer, salivarygland cancer, lung cancer, renal cancer, colon cancer, colorectalcancer, thyroid cancer, bladder cancer, glioma, melanoma, metastaticbreast cancer, non-small cell lung cancer, epidermoid carcinoma,fibrosarcoma, melanoma, nasopharyngeal carcinoma, and squamous cellcarcinoma. In some preferred embodiments, U1-49, U1-53 or U1-59 can beuse in the treatment of patients with cancers including ovarian cancerwhose disease has progressed or recurred after platinum-basedchemotherapy, in combination with doxorubicin HCl liposome injection(Doxil).

Irinotecan hydrochloride hydrate (irinotecan:(+)-(4S)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino)carbonyloxy]-1H-pyrano[3′,4′:6,7]indolizino[1-2-b]quinoline-3,14(4H,12H)-dionehydrochloride trihydrate) (Yakult, EP Publication Nos. 137145 and 56692)is marketed as Campto, Camptosar and Ircan. In some embodiments, acombination treatment for HER3-associated disease can includeadministering U1-49, U1-53 or U1-59 in combination with a second agentas described herein and irinotecan hydrochloride hydrate, or U1-49,U1-53 or U1-59 in combination with a second agent as described herein,irinotecan hydrochloride hydrate, and one or more other agent(s) such as5-FU(5′-deoxy-5-fluorouridine or 5-fluoro-2,4(1H,3H)-pyrimidinedione),calcium folinate (N-[1-[[(2-amino-5-formyl-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methylamino]benzoyl]-L-glutamic acidcalcium salt (1:1)) or calcium levofolinate ((−)-calciumN-[4-[[[(6S)-2-amino-5-formyl-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl]methyl]amino]benzoyl]-L-glutamate),and combinations thereof, for treatment of cancer such as breast cancer,gastrointestinal cancer, pancreatic cancer, prostate cancer, ovariancancer, stomach cancer, endometrial cancer, salivary gland cancer, lungcancer, renal cancer, colon cancer, colorectal cancer, thyroid cancer,bladder cancer, glioma, melanoma, metastatic breast cancer, non-smallcell lung cancer, epidermoid carcinoma, fibrosarcoma, melanoma,nasopharyngeal carcinoma, and squamous cell carcinoma.

In some preferred embodiments, U1-49, U1-53 or U1-59 can be use in thetreatment of patients with cancers including colorectal cancer andmetastatic colorectal cancer after failure of 5-fluorouracil-basedchemotherapy, in combination with 5-fluorouracil-based chemotherapy. Insome further embodiments, U1-49, U1-53 or U1-59 can be use in thetreatment of the treatment of patients with cancers including colorectalcancer and metastatic colorectal cancer with wild-type K-RAS afterfailure of 5-fluorouracil-based chemotherapy, in combination withcetuximab and irinotecan.

In some embodiments, the additional agents to be use in the compositionsand methods disclosed herein, which are exchangeable with the secondagent as disclosed herein, may be an artificial or naturally-occurringscaffold which is not an antibody, but has an antibody-like activity(e.g., has an activity similar to that of an antibody).

In some other embodiments, said additional agents, which areexchangeable with the second agent disclosed herein, can be agentsinhibit, block or reduce (act as antagonists towards), or, activate,stimulate or accelerate (act as agonist towards) an activity of othertargets, including but not limited to those affect cellular growthand/or survival pathways, such as PI3K inhibitors, AKT inhibitors, mTORinhibitors, RAF/B-RAF inhibitors, RAS inhibitors, MEK inhibitors, DeathReceptor inhibitors including DR4 and DR5 agonists such as anti-DR4 orDR5 agonistic antibodies (for example, cedelizumab, tigatuzumab,drozirumab, conatumumab), PPAR gamma agonists (for example, efatutazone,troglitazone, pioglitazone, rosiglitazone), c-MET inhibitors, Hsp-90inhibitors and telomerase inhibitors.

In some other embodiments, said additional agents, which areexchangeable with the second agent as disclosed herein, can beanti-angiogenics, including but not limited to, VEGFantagonists/inhibitors (for example, bevacizumab, vandetanib).

In some further embodiments, said additional agents, which areexchangeable with the second agent, can be immunotherapeutic such asvaccines or cellular therapeutics.

As further described below, these and other agents can be containedwithin the compositions provided herein, and can be administered in avariety of different forms, combinations and dosages.

5. Compositions

This document also provides pharmaceutical compositions comprising aHER-3 binding agent as described herein, in combination with a secondagent that is directed against another HER family protein or is achemotherapeutic compound, as well as one or more pharmaceuticallyacceptable carriers, diluents and/or adjuvants. The term “pharmaceuticalcomposition,” as used herein, refers to a chemical compound orcomposition capable of inducing a desired therapeutic effect whenproperly administered to a patient (The McGraw-Hill Dictionary ofChemical Terms, Parker, Ed., McGraw-Hill, San Francisco (1985)). Thepotency of the pharmaceutical compositions provided herein typically isbased on the binding of the at least one binding protein to HER-3. Insome embodiments, this binding can lead to a reduction of theHER-3-mediated signal transduction.

A “pharmaceutically acceptable carrier” (also referred to herein as an“excipient” or a “carrier”) is a pharmaceutically acceptable solvent,suspending agent, stabilizing agent, or any other pharmacologicallyinert vehicle for delivering one or more therapeutic compounds (e.g.,HER binding proteins) to a subject, which is nontoxic to the cell ormammal being exposed thereto at the dosages and concentrations employed.Pharmaceutically acceptable carriers can be liquid or solid, and can beselected with the planned manner of administration in mind so as toprovide for the desired bulk, consistency, and other pertinent transportand chemical properties, when combined with one or more of therapeuticcompounds and any other components of a given pharmaceuticalcomposition. Typical pharmaceutically acceptable carriers that do notdeleteriously react with amino acids include, by way of example and notlimitation: water, saline solution, binding agents (e.g.,polyvinylpyrrolidone or hydroxypropyl methylcellulose), fillers (e.g.,lactose and other sugars, gelatin, or calcium sulfate), lubricants(e.g., starch, polyethylene glycol, or sodium acetate), disintegrates(e.g., starch or sodium starch glycolate), and wetting agents (e.g.,sodium lauryl sulfate). Pharmaceutically acceptable carriers alsoinclude aqueous pH buffered solutions or liposomes (small vesiclescomposed of various types of lipids, phospholipids and/or surfactantswhich are useful for delivery of a drug to a mammal). Further examplesof pharmaceutically acceptable carriers include buffers such asphosphate, citrate, and other organic acids, antioxidants such asascorbic acid, low molecular weight (less than about 10 residues)polypeptides, proteins such as serum albumin, gelatin, orimmunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone,amino acids such as glycine, glutamine, asparagine, arginine or lysine,monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose or dextrins, chelating agents such as EDTA, sugaralcohols such as mannitol or sorbitol, salt-forming counterions such assodium, and/or nonionic surfactants such as TWEEN™, polyethylene glycol(PEG), and PLURONICS™.

Liposomes are vesicles that have a membrane formed from a lipophilicmaterial and an aqueous interior that can contain the composition to bedelivered. Liposomes can be particularly useful due to their specificityand the duration of action they offer from the standpoint of drugdelivery. Liposome compositions can be formed, for example, fromphosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoyl phosphatidylglycerol, or dioleoylphosphatidylethanolamine. Numerous lipophilic agents are commerciallyavailable, including LIPOFECTIN® (Invitrogen/Life Technologies,Carlsbad, Calif.) and EFFECTENE™ (Qiagen, Valencia, Calif.).

In some embodiments, at least one of the agents contained in apharmaceutical composition (e.g., a HER-3 binding agent or an agent thatbinds and/or inhibits another HER family member) can be coupled to aneffector such as calicheamicin, duocarmycins, auristatins,maytansinoids, a radioisotope, or a toxic chemotherapeutic agent such asgeldanamycin and maytansine. Such conjugates can be particularly usefulfor targeting cells (e.g., cancer cells) expressing HER-3.

Linking binding proteins to radioisotopes can provide advantages totumor treatments. Unlike chemotherapy and other forms of cancertreatment, radioimmunotherapy or the administration of aradioisotope-binding protein combination can directly target cancercells with minimal damage to surrounding normal, healthy tissue. Withthis “magic bullet,” patients can be treated with much smallerquantities of radioisotopes than other forms of treatment availabletoday. Suitable radioisotopes include, for example, yttrium⁹⁰ (⁹⁰Y),indium¹¹¹ (¹¹¹In), ¹³¹I, ⁹⁹mTc, radiosilver-111, radiosilver-199, andBismuth²¹³. The linkage of radioisotopes to binding proteins may beperformed with, for example, conventional bifunctional chelates. Sincesilver is monovalent, for radiosilver-111 and radiosilver-199 linkage,sulphur-based linkers may be used (Hazra et al. (1994) Cell Biophys.24-25:1-7). Linkage of silver radioisotopes may involve reducing theimmunoglobulin with ascorbic acid. Furthermore, tiuxetan is an MX-DTPAlinker chelator attached to ibritumomab to form ibritumomab tiuxetan(Zevalin) (Witzig (2001) Cancer Chemother. Pharmacol. 48 (Suppl1):91-95). Ibritumomab tiuxetan can react with radioisotypes such asindium¹¹¹ (¹¹¹In) or ⁹⁰Y to form ¹¹¹In-ibritumomab tiuxetan and⁹⁰Y-ibritumomab tiuxetan, respectively.

The binding proteins described herein, particularly when used to treatcancer, can be conjugated with toxic chemotherapeutic drugs such asmaytansinoids, (Hamann et al. (2002) Bioconjug. Chem. 13:40-46),geldanamycinoids (Mandler et al. (2000) J. Natl. Cancer Inst.92:1549-1551) and maytansinoids, for example, the maytansinoid drug, DM1(Liu et al. (1996) Proc. Natl. Acad. Sci. US 93:8618-8623). Linkers thatrelease the drugs under acidic or reducing conditions or upon exposureto specific proteases may be employed with this technology. A bindingprotein may be conjugated as described in the art.

In some embodiments, a binding protein can be conjugated toauristatin-PE. Auristatin-PE, e.g., is an antimicrotubule agent that isa structural modification of the marine, shell-less mollusk peptideconstituent dolastatin 10. Auristatin-PE has both anti-tumor activityand anti-tumor vascular activity (Otani et al. (2000) Jpn. J. CancerRes. 91:837-44). For example, auristatin-PE inhibits cell growth andinduces cell cycle arrest and apoptosis in pancreatic cancer cell lines(Li et al. (1999) Int. J. Mol. Med. 3:647-53). Accordingly, tospecifically target the anti-tumor activity and anti-tumor vascularactivities of auristatin-PE to particular tumors, auristatin-PE may beconjugated to a binding protein as provided herein.

The pharmaceutical compositions provided herein also can contain atleast one further active agent. Examples of further active agentsinclude antibodies or low molecular weight inhibitors of other receptorprotein kinases, such as IGFR-1 and c-met, receptor ligands such asvascular endothelial factor (VEGF), cytotoxic agents such asdoxorubicin, cisplatin or carboplatin, cytokines, or anti-neoplasticagents. Many anti-neoplastic agents are known in the art. In someembodiments, an anti-neoplastic agent can be selected from the group oftherapeutic proteins including, but not limited to, antibodies andimmunomodulatory proteins. In some embodiments, an anti-neoplastic agentcan be selected from the group of small molecule inhibitors andchemotherapeutic agents consisting of mitotic inhibitors, kinaseinhibitors, alkylating agents, anti-metabolites, intercalatingantibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes,topoisomerase inhibitors, histone deacetylase inhibitors, anti-survivalagents, biological response modifiers, anti-hormones (e.g.,anti-androgens), microtubule stimulants, anthracyclins, andanti-angiogenesis agents. When the anti-neoplastic agent is radiation,treatment can be achieved either with an internal source (e.g.,brachytherapy) or an external source (e.g., external beam radiationtherapy). The one or more further active agent(s) can be administeredwith the HER3-binding agent and the second agent either simultaneouslyor separately, in a single formulation or in individual (separate)formulations for each active agent.

The pharmaceutical compositions provided herein can be especially usefulfor diagnosis, prevention, or treatment of a hyperproliferative disease.The hyperproliferative disease can be associated with increased HERfamily signal transduction. In particular, the disease can be associatedwith increased HER-3 phosphorylation, increased complex formationbetween HER-3 and other members of the HER family, increased PI₃ kinaseactivity, increased c-jun terminal kinase activity and/or AKT activity,increased ERK2 and/or PYK2 activity, or any combination thereof. Thehyperproliferative disease can be, for example, selected from the groupconsisting of breast cancer, gastrointestinal cancer, pancreatic cancer,prostate cancer, ovarian cancer, stomach cancer, endometrial cancer,salivary gland cancer, lung cancer, kidney cancer, colon cancer,colorectal cancer, thyroid cancer, bladder cancer, glioma, melanoma, orother HER-3 expressing or overexpressing cancers, and the formation oftumor metastases.

Pharmaceutical compositions can be formulated by mixing one or moreactive agents with one or more physiologically acceptable carriers,diluents, and/or adjuvants, and optionally other agents that are usuallyincorporated into formulations to provide improved transfer, delivery,tolerance, and the like. A pharmaceutical composition can be formulated,e.g., in lyophilized formulations, aqueous solutions, dispersions, orsolid preparations, such as tablets, dragees or capsules. A multitude ofappropriate formulations can be found in the formulary known to allpharmaceutical chemists: Remington's Pharmaceutical Sciences (18^(th)ed, Mack Publishing Company, Easton, Pa. (1990)), particularly Chapter87 by Block, Lawrence, therein. These formulations include, for example,powders, pastes, ointments, jellies, waxes, oils, lipids, lipid(cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNAconjugates, anhydrous absorption pastes, oil-in-water and water-in-oilemulsions, emulsions carbowax (polyethylene glycols of various molecularweights), semi-solid gels, and semi-solid mixtures containing carbowax.Any of the foregoing mixtures may be appropriate in treatments andtherapies as described herein, provided that the active agent in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See, also, Baldrick (2000) Regul. Toxicol. Pharmacol.32:210-218; Wang (2000) Int. J. Pharm. 203:1-60; Charman (2000) J.Pharm. Sci. 89:967-978; and Powell et al. (1998) PDA J. Pharm. Sci.Technol. 52:238-311), and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

This document also pertains to the use of at least one agent (e.g., anisolated HER-3 binding protein) as described herein, and at least oneother active agent (e.g., an agent that binds to another HER familymember or a chemotherapeutic compound) in admixture withpharmaceutically acceptable carriers, diluents and/or adjuvants, for themanufacture of a pharmaceutical composition for diagnosis, prevention ortreatment of a hyperproliferative disease (e.g., a disease associatedwith HER-3). The pharmaceutical composition can be a pharmaceuticalcomposition as described herein, and the hyperproliferative disease canbe a hyperproliferative disease as described herein.

Methods for formulating and subsequently administering therapeuticcompositions are well known to those skilled in the art. Dosinggenerally is dependent on the severity and responsiveness of the diseasestate to be treated, with the course of treatment lasting from severaldays to several months, or until a cure is effected or a diminution ofthe disease state is achieved. Persons of ordinary skill in the artroutinely determine optimum dosages, dosing methodologies and repetitionrates. Optimum dosages can vary depending on the relative potency ofindividual polypeptides, and can generally be estimated based on EC₅₀found to be effective in in vitro and in vivo animal models. Typically,dosage is from 0.1 μg to 100 mg per kg of body weight, and may be givenonce or more daily, biweekly, weekly, monthly, or even less often.Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate.

Pharmaceutical compositions can be administered by a number of methods,depending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration can be, for example, topical(e.g., transdermal, sublingual, ophthalmic, or intranasal); pulmonary(e.g., by inhalation or insufflation of powders or aerosols); oral; orparenteral (e.g., by subcutaneous, intrathecal, intraventricular,intramuscular, or intraperitoneal injection, or by intravenous drip).Administration can be rapid (e.g., by injection) or can occur over aperiod of time (e.g., by slow infusion or administration of slow releaseformulations). For treating tissues in the central nervous system, HER-3binding proteins can be administered by injection or infusion into thecerebrospinal fluid, typically with one or more agents capable ofpromoting penetration of the polypeptides across the blood-brainbarrier.

Compositions and formulations for parenteral, intrathecal orintraventricular administration can include sterile aqueous solutions,which also can contain buffers, diluents and other suitable additives(e.g., penetration enhancers, carrier compounds and otherpharmaceutically acceptable carriers).

Pharmaceutical compositions include, without limitation, solutions,emulsions, aqueous suspensions, and liposome-containing formulations.These compositions can be generated from a variety of components thatinclude, for example, preformed liquids, self-emulsifying solids andself-emulsifying semisolids. Emulsions are often biphasic systemscomprising of two immiscible liquid phases intimately mixed anddispersed with each other; in general, emulsions are either of thewater-in-oil (w/o) or oil-in-water (o/w) variety. Emulsion formulationshave been widely used for oral delivery of therapeutics due to theirease of formulation and efficacy of solubilization, absorption, andbioavailability.

HER binding agents can further encompass any pharmaceutically acceptablesalts, esters, or salts of such esters, or any other compound which,upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, this document providespharmaceutically acceptable salts of small molecules and polypeptides,prodrugs and pharmaceutically acceptable salts of such prodrugs, andother bioequivalents. The term “prodrug” indicates a therapeutic agentthat is prepared in an inactive form and is converted to an active form(i.e., drug) within the body or cells thereof by the action ofendogenous enzymes or other chemicals and/or conditions. The term“pharmaceutically acceptable salts” refers to physiologically andpharmaceutically acceptable salts of the polypeptides provided herein(i.e., salts that retain the desired biological activity of the parentpolypeptide without imparting undesired toxicological effects). Examplesof pharmaceutically acceptable salts include, but are not limited to,salts formed with cations (e.g., sodium, potassium, calcium, orpolyamines such as spermine); acid addition salts formed with inorganicacids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, or nitric acid); and salts formed with organic acids(e.g., acetic acid, citric acid, oxalic acid, palmitic acid, or fumaricacid).

Some embodiments provided herein include pharmaceutical compositionscontaining (a) one or more HER-3 binding agents; (b) one or more agentsthat bind to another HER family member; and (c) one or more other agentsthat function by a different mechanism. For example, one or more agentsof (c) are exchangeable with those of (b); anti-inflammatory drugs,including but not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, can be included incompositions. Other non-polypeptide agents (e.g., chemotherapeuticagents) also are within the scope of this document. Such combinedcompounds can be used together or sequentially.

Compositions additionally can contain other adjunct componentsconventionally found in pharmaceutical compositions. Thus, thecompositions also can include compatible, pharmaceutically activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or additional materials usefulin physically formulating various dosage forms of the compositionsprovided herein, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers.Furthermore, the composition can be mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavorings,and aromatic substances. When added, however, such materials should notunduly interfere with the biological activities of the polypeptidecomponents within the compositions provided herein. The formulations canbe sterilized if desired.

The pharmaceutical formulations, which can be presented conveniently inunit dosage faun, can be prepared according to conventional techniqueswell known in the pharmaceutical industry. Such techniques include thestep of bringing into association the active ingredients (e.g., the HERfamily binding agents provided herein) with the desired pharmaceuticalcarrier(s) or excipient(s). Typically, the formulations can be preparedby uniformly and bringing the active ingredients into intimateassociation with liquid carriers or finely divided solid carriers orboth, and then, if necessary, shaping the product. Formulations can besterilized if desired, provided that the method of sterilization doesnot interfere with the effectiveness of the polypeptide contained in theformulation.

The compositions described herein can be formulated into any of manypossible dosage forms such as, but not limited to, tablets, capsules,liquid syrups, soft gels, suppositories, and enemas. The compositionsalso can be formulated as suspensions in aqueous, non-aqueous or mixedmedia. Aqueous suspensions further can contain substances that increasethe viscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol, and/or dextran. Suspensions also cancontain stabilizers.

HER binding proteins can be combined with packaging material and sold askits for treating HER-3 associated diseases. Components and methods forproducing articles of manufacture are well known. The articles ofmanufacture may combine one or more of the polypeptides and compoundsset out in the above sections. In addition, the article of manufacturefurther may include, for example, buffers or other control reagents forreducing or monitoring reduced immune complex formation. Instructionsdescribing how the polypeptides are effective for treating HER-3associated diseases can be included in such kits. Any of the firstagents, the second agents and additional agents could be delivered innanoparticle(s) or liposome(s), or any other suitable form(s)

6. Methods

This document also provides methods for treating or preventing diseasesand conditions associated with expression of HER-3. For example, amethod can include contacting a subject or a biological sample from asubject (e.g., a mammal such as a human) with a HER-3 binding protein incombination with a second agent as described herein. The sample may be acell that shows expression of HER-3, such as a tumor cell, a bloodsample or another suitable sample. In some embodiments, the contactingoccurs in vivo, such as when a composition containing a HER-3 bindingagent and a second agent that binds to another member of the HER familyis administered to a subject in need thereof. The diseases or conditionsassociated with expression of HER-3 that can be treated using themethods described herein include, for example, hyperproliferativediseases such as breast cancer, gastrointestinal cancer, pancreaticcancer, prostate cancer, ovarian cancer, stomach cancer, endometrialcancer, salivary gland cancer, lung cancer, kidney cancer, colon cancer,colorectal cancer, thyroid cancer, bladder cancer, glioma, melanoma,renal cancer, metastatic breast cancer, non-small cell lung cancer,epidermoid carcinoma, fibrosarcoma, melanoma, nasopharyngeal carcinoma,squamous cell carcinoma, and other HER-3-positive, -expressing or-overexpressing cancers.

The term “treatment or prevention,” when used herein, refers to boththerapeutic treatment and prophylactic or preventative measures, whichcan be used to prevent, slow, or lessen the effects of the targetedpathologic condition or disorder. Those in need of prevention ortreatment can include those already having the disorder, as well asthose who may be likely to develop the disorder, or those in whom thedisorder is to be prevented. The patient in need of prevention ortreatment can be a mammalian patient (i.e., any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc.) In some embodiments, the patient in need of treatment isa human patient.

Methods for preventing or treating diseases or conditions associatedwith expression of HER-3 in a patient in need thereof can includeadministering to the patient effective amounts of at least one HER-3binding agent as described herein and at least one other agent againstanother HER family member, or a chemotherapeutic compound (e.g., atleast one of the “additional/further” agents described above, which areexchangeable with the second agents binding to and/or inhibiting anotherHER family). Such treatment can, for example, inhibit abnormal cellgrowth, migration or invasion. The agent against HER-3 and the at leastone other agent can be administered simultaneously (e.g., when they arecontained in the same composition, or by admixture into a common i.v.bag), or separately (e.g., sequentially). The diseases or conditionsassociated with the expression of HER-3 that can be treated using themethods provided herein include, for example, the hyperproliferativediseases listed herein. The patient in need of prevention or treatmentcan be a mammal (e.g., a human, a domestic or farm animal, or a zoo,sport, or pet animal such as a dog, cat, cow, horse, sheep, pig, goat,or rabbit). In some cases, the patient is a human patient.

As used herein, the term “effective amount” is an amount of an agentthat results in a decrease or stabilization in one or more symptoms orclinical characteristics of the HER-3 associated condition beingtreated. For example, administration of an effective amount of acomposition as described herein can result in slowing of tumor growthprogression, in decreased tumor size, or in decreased activation ofHER-3 or HER-3-responsive biomarkers (e.g., Akt, HER-2, ERK, or EGF-R).The slowing or decrease can be any reduction as compared to a previousvalue (e.g., a 5%, 10%, 20%, 25%, or more than 25% reduction in symptomor characteristic). In some embodiments, an “effective amount” canresult in stable disease.

In addition to classical modes of administration of potential bindingprotein therapeutics, e.g., via the above mentioned formulations, newlydeveloped modalities of administration may also be useful. For example,local administration of ¹³¹I-labeled monoclonal antibody for treatmentof primary brain tumors after surgical resection has been reported.Additionally, direct stereotactic intracerebral injection of monoclonalantibodies and their fragments is also being studied clinically andpre-clinically. Intracarotid hyperosmolar perfusion is an experimentalstrategy to target primary brain malignancy with drug conjugated humanmonoclonal antibodies.

As described above, the dose of the agents administered can depend on avariety of factors. These include, for example, the nature of theagents, the tumor type, and the route of administration. It should beemphasized that the present methods are not limited to any particulardoses. Methods for determining suitable doses are known in the art, andinclude those described in the Examples herein.

Depending on the type and severity of the condition to be treated, up toabout 20 mg/kg of each HER binding antibody can be administered to apatient in need thereof, e.g., by one or more separate administrationsor by continuous infusion. A typical daily dosage might range from about1 μg/day to about 100 mg/day or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition to be treated, the treatment can be sustaineduntil a desired suppression of disease symptoms occurs.

In some embodiments, a method as provided herein can include analyzing aparticular marker (e.g., HER-3) in a biological sample from a subject todetermine whether the subject has a disease associated with HER-3expression. Such methods can be used to select subjects having diseasesassociated with HER-3. In such methods, the analyzing step can be doneprior to the step of administration, as such screening of patients mayavoid treatments that are not likely to be effective. Thus, in somecases, the methods provided herein can further include detecting HER-3antigen in or on a cell, for determination of HER-3 antigenconcentration in patients suffering from a hyperproliferative disease asmentioned above, or for staging of a hyperproliferative disease in apatient. In order to stage the progression of a hyperproliferativedisease in a subject under study, or to characterize the response of thesubject to a course of therapy, a sample of blood can be taken from thesubject and the concentration of the HER-3 antigen present in the samplecan be determined. The concentration so obtained can be used to identifyin which range of concentrations the value falls. The range soidentified can be correlated with a stage of progression or a stage oftherapy identified in the various populations of diagnosed subjects,thereby providing a stage for the subject under study. A biopsy of thedisease, e.g., cancerous, tissue obtained from the patient also can beused assess the amount of HER-3 antigen present. The amount of HER-3antigen present in the disease tissue may be assessed using, forexample, immunohistochemistry, ELISA, or antibody array using HER-3antibodies as described herein. Other parameters of diagnostic interestare the dimerization state as well as the dimerization partners of theHER-3 protein and the activation state of it and its partners. Proteinanalytical methods to determine those parameters are well known in theart and are among others western blot and immunoprecipitationtechniques, FACS analysis, chemical crosslinking, bioluminescenceresonance energy transfer (BRET), fluorescence resonance energy transfer(FRET) and the like (e.g., Price et al. (2002) Methods Mol. Biol.218:255-268, or eTag technology (WO 05/03707, WO 04/091384, and WO04/011900).

In some cases, a method as provided herein can include one or more stepsfor monitoring the therapeutic outcome of the treatment. For example, asubject can be monitored for symptoms of their disease, to determinewhether a reduction in symptoms has occurred. The subject also can bemonitored, for example, for potential side effects of the treatment. Themonitoring can be done after the administration step, and, in someembodiments, can be done multiple times (e.g., between administrations,if dosages are given more than once). Such methods can be used to assessefficacy and safety of the treatment methods described herein, forexample.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 HER-3 Antigen and Cell Line Preparation

Recombinant HER-3 proteins were prepared. The extracellular domain ofHER-3 (ECD) cDNA was cloned by polymerase chain reaction (PCR) frompcDNA3-HER-3 (expression vector with full length human HER-3, Wallaschet al. (1995) EMBO J. 14:4267-4275) with primers based on the sequenceof HER-3 (GeneBank Accession No. NM_001982): Forward primer:5′-CGGGATCCATGTCCTAGCCTAGGGGC-3′ (SEQ ID NO: 233); Reverse primer:5′-GCTCTAGATTAATGATGATGATGATGATGTTGTCCTAAACAGTCTTG-3′ (SEQ ID NO: 234).

The PCR product was digested with BamH1 and XbaI and ligated into pcDNA3(Invitrogen) digested with BamH1 and XbaI. Plasmids were transfectedinto HEK293 cells using a CaPO₄ method. The HER-3-HIS fusion protein waspurified from harvested conditioned media via Ni-NTA affinitychromatography.

RatI HER-3 cells were generated by retroviral gene transfer. Briefly,GP+E 86 cells (3×10⁵) were seeded on a 60 mm culture disc andtransfected with 2 μg/ml plXSN vector or plXSN-HER-3 cDNA (C. Wallasch,PhD Thesis, Max-Planck Institute of Biochemistry, Martinsried, Germany)using the calcium phosphate method. After 24 hours, the medium wasreplaced with fresh medium and the GP+E 86 cells were incubated for 4-8hours. Subconfluent Rat1 cells (2×10⁵ cells per 6 cm dish) were thenincubated with supernatants of GP+E 86 cells releasing high titer pLXSNor pLXSN-HER-3, p virus (>1×10⁶ G418 c.f.u./ml; m.o.i. of 10) for 4-12hours in the presence of Polybrene (4 mg/ml; Aldrich). After changingthe medium, selection of RatI cells with G418 was started. Usually,stable clones were picked after selection for 21 days.

Example 2 HER-3 Expression in Human Cancer Cell Lines

HER-3 expression was quantified in a panel of human cancer cell lines toelucidate the role of HER-3 in human cancer formation. Cancer cell lineswere grown as recommended by the ATCC. In detail, 10⁵ cells wereharvested with 10 mM EDTA in PBS, washed once with FACS buffer (PBS, 3%FCS, 0.4% azide) and seeded on a 96-well round bottom plate. The cellswere spun for 3 minutes at 1000 rpm to remove supernatant and thenresuspended with HER-3 antibody 2D1D12 (WO03013602) (3 μg/ml). Cellsuspensions were incubated on ice for 1 hour, washed twice with FACSbuffer, and resuspended with secondary antibody (100 μl/well)donkey-anti-human-PE (Jackson) diluted 1:50 in FACS buffer. The cellsuspensions were incubated on ice and in the dark for 30 minutes, washedtwice with FACS buffer and analyzed (FACS, Beckman Coulter). HER-3 wasexpressed in a variety of human cancer cell lines, including variousbreast, colon, epidermoid, melanoma, nasopharynx, ovarian, pancreas, andprostate cell lines. See, the figures of US Publication No. 20080124345,which are hereby incorporated herein by reference in their entirety.

Example 3 Immunization and Titering

The HER-3 ECD protein that was prepared as described in Example 1 andC32 cells (Human melanoma; ATCC #CRL-1585) were used as antigen.Monoclonal antibodies against HER-3 were developed by sequentiallyimmunizing XENOMOUSE® mice (strains XMG1 and XMG4; Abgenix, Inc.,Fremont, Calif.). XENOMOUSE® animals were immunized via the footpad forall injections. The total volume of each injection was 50 μl per mouse,25 μl per footpad.

For cohort #1 (10 XMG1 mice), the initial immunization was with 10 μg ofHER-3 ECD protein admixed 1:1 (v/v) with TITERMAX GOLD® (Sigma,Oakville, ON) per mouse. The subsequent five boosts were made with 10 μgof HER-3 ECD protein admixed 1:1 (v/v) with 100 μg alum gel (Sigma,Oakville, ON) in pyrogen-free D-PBS. The sixth boost consisted of 10 μgof HER-3 ECD protein admixed 1:1 (v/v) with TITERMAX GOLD®. The seventhinjection consisted of 10 μg of HER-3 ECD protein admixed 1:1 v/v with100 μg alum gel. A final boost was made with 10 μg HER-3 ECD protein inpyrogen-free DPBS, without adjuvant. The XENOMOUSE® mice were immunizedon days 0, 4, 7, 11, 15, 20, 24, and 29 for this protocol, and fusionswere performed on day 33. The two bleeds were made through Retro-OrbitalBleed procedure on day 13 after the fourth boost and on day 19 after thesixth boost. There was no cohort #2. For Cohort #3 (10 XMG1 mice) andCohort #4 (10 XMG4 mice), the first injection was with 10⁷ C32 cells inpyrogen-free Dulbecco's PBS (DPBS) admixed 1:1 (v/v) with TITERMAX GOLD®per mouse. The next four boosts were with 10⁷ C32 cells in pyrogen-freeDPBS, admixed with 25 μg of Adju-Phos and 10 μg CpG per mouse. The sixthboost was with 10⁷ C32 cells in pyrogen-free DPBS, admixed 1:1 (v/v)with TITERMAX GOLD® per mouse. The seventh, eighth, and ninth boostswere with 10⁷ C32 cells in pyrogen-free DPBS, admixed with 25 μg ofAdju-Phos and 10 μg CpG per mouse. The tenth to fourteenth boosts werewith 5 μg of HER-3 ECD protein in pyrogen-free DPBS, admixed with 25 μgof Adju-Phos and 10 μg CpG per mouse. A final boost consisted of 5 μg ofHER-3 ECD protein in pyrogen-free DPBS, without adjuvant. For bothCohorts #3 and #4, the mice were immunized on days 0, 3, 7, 11, 14, 17,21, 24, 28, 33, 35, 38, 42 and 45, and fusions were performed on day 49.The three bleeds were made through Retro-Orbital Bleed procedure on day12 after the fourth boost, on day 19 after the sixth boost, and on day40 after twelfth boost.

Selection of animals for harvest by titer: For cohort #1, anti-HER-3antibody titers in the serum from immunized mice were determined byELISA against HER-3 ECD protein. The specific titer of each XENOMOUSE®animal was determined from the optical density at 650 nm, and is shownin TABLE 1 below. The titer value is the reciprocal of the greatestdilution of sera with an OD reading two-fold that of background.Therefore, the higher the number, the greater the humoral immuneresponse to HER-3 ECD.

TABLE 1 Cohort #1, XMG1 Mouse ID After 4 injections After 6 injectionsP3421 8,000 11,000 P3422 850 2,600 P3423 2,700 5,200 P3424 3,200 9,100P3425 5,400 2,500 P3426 700 1,500 P3427 5,800 7,000 P3428 3,900 4,300P3429 2,200 2,500 P34210 600 850 NC 250 175 PC 377,000 311,000 NC mAbIL-8, D39.2.1 PC xHER-3-2D1D12

For cohorts #3 and #4, anti-HER-3 antibody titers in the serum fromimmunized mice were determined by FACS using Rat1/HER-3 (antigenpositive cell line) cells and Rat1/pLSXN (antigen negative cell line)cells. Data are shown in TABLES 2 and 3, and are presented as geometricmean (GeoMean) fluorescent intensity of cell anti-HER-3 cell staining byserial dilutions of serum samples.

TABLE 2 Cohort #3, XMG1 After 6 injections After 12 injections pos cellsneg cells pos cells neg cells Mouse ID Sample GeoMean GeoMean GeoMeanGeoMean Q832-1 1:50  9 10 11 10 1:250  6 9 6 6 1:1250 6 7 4 4 Q832-21:50  8 10 29 42 1:250  7 8 11 11 1:1250 5 6 6 5 Q832-3 1:50  7 12 11 91:250  5 7 5 5 1:1250 5 5 4 4 Q832-4 1:50  6 10 9 9 1:250  6 6 5 51:1250 5 5 4 4 Q832-5 1:50  11 11 17 13 1:250  10 9 7 6 1:1250 6 8 5 4Q832-6 1:50  7 11 15 14 1:250  7 7 7 6 1:1250 5 6 6 4 Q832-7 1:50  8 117 15 1:250  6 7 5 5 1:1250 5 5 4 4 Q832-8 1:50  7 8 11 20 1:250  6 6 7 81:1250 5 5 5 4 Q832-9 1:50  7 12 15 16 1:250  6 8 6 5 1:1250 6 6 4 4Q832-10 1:50  8 13 34 38 1:250  6 8 9 8 1:1250 6 6 5 4

TABLE 3 Cohort #4, XMG4 After 6 injections After 12 injections pos cellsneg cells pos cells neg cells Mouse Sample GeoMean GeoMean GeoMeanGeoMean Q856-1 1:50  4 6 91 44 1:250  4 5 32 18 1:1250 4 4 19 10 Q856-21:50  4 8 148 54 1:250  4 5 89 23 1:1250 4 4 42 9 Q856-3 1:50  4 5 72 141:250  4 4 28 6 1:1250 4 4 18 4 Q856-4 1:50  4 5 11 49 1:250  4 5 10 171:1250 4 4 8 7 Q856-5 1:50  4 4 74 20 1:250  4 4 30 14 1:1250 4 4 16 6Q856-6 1:50  4 5 86 21 1:250  4 4 32 10 1:1250 4 4 16 5 Q856-7 1:50  5 674 32 1:250  4 5 32 14 1:1250 4 4 16 6 Q856-8 1:50  4 5 106 14 1:250  44 45 6 1:1250 4 4 22 4 Q856-9 1:50  5 6 53 22 1:250  4 4 17 11 1:1250 44 11 5 Q856-10 1:50  4 5 72 53 1:250  4 4 26 17 1:1250 4 4 15 7

Example 4 Recovery of Lymphocytes, B-Cell Isolations, Fusions andGeneration of Hybridomas

Immunized mice were sacrificed and the lymph nodes were harvested andpooled from each cohort. The lymphoid cells were dissociated by grindingin DMEM to release the cells from the tissues, and the cells weresuspended in DMEM. The cells were counted, and 0.9 ml DMEM per 100million lymphocytes was added to the cell pellet to resuspend the cellsgently but completely. Using 100 μl of CD90+ magnetic beads per 100million cells, the cells were labeled by incubating the cells with themagnetic beads at 4° C. for 15 minutes. The magnetically-labeled cellsuspension containing up to 10⁸ positive cells (or up to 2×10⁹ totalcells) was loaded onto a LS+ column and the column washed with DMEM. Thetotal effluent was collected as the CD90-negative fraction (most ofthese cells were expected to be B cells).

The fusion was performed by mixing washed enriched B cells from aboveand nonsecretory myeloma P3X63Ag8.653 cells purchased from ATCC (Cat.No. CRL 1580) (Kearney et al. (1979) J. Immunol. 123:1548-1550) at aratio of 1:1. The cell mixture was gently pelleted by centrifugation at800 g. After complete removal of the supernatant, the cells were treatedwith 2 to 4 ml of pronase solution (CalBiochem, Cat. No. 53702; 0.5mg/ml in PBS) for no more than 2 minutes. Then 3 to 5 ml of FBS wasadded to stop the enzyme activity, and the suspension was adjusted to 40ml total volume using electro cell fusion solution, ECFS (0.3 M sucrose,Sigma, Cat. No. 57903, 0.1 mM magnesium acetate, Sigma, Cat. No. M2545,0.1 mM calcium acetate, Sigma, Cat. No. C4705). The supernatant wasremoved after centrifugation and the cells were resuspended in 40 mlECFS. This wash step was repeated and the cells again were resuspendedin ECFS to a concentration of 2×10⁶ cells/ml.

Electro-cell fusion was performed using a fusion generator, modelECM2001, Genetronic, Inc., San Diego, Calif. The fusion chamber size was2.0 ml, and the following instrument settings were used: Alignmentconditions: voltage: 50 V, time: 50 seconds; membrane breaking: voltage:3000 V, time: 30 μseconds; post-fusion holding time: 3 seconds.

After ECF, the cell suspensions were removed from the fusion chamberunder sterile conditions and transferred into a sterile tube containingthe same volume of Hybridoma Culture Medium (DMEM (JRH Biosciences), 15%FBS (Hyclone), supplemented with L-glutamine, pen/strep, OPI(oxaloacetate, pyruvate, bovine insulin) (all from Sigma) and IL-6(Boehringer Mannheim). The cells were incubated for 15 to 30 minutes at37° C., and then centrifuged at 400 g for five minutes. The cells weregently resuspended in a small volume of Hybridoma Selection Medium(Hybridoma Culture Medium supplemented with 0.5× HA (Sigma, Cat. No.A9666)), and the volume was adjusted appropriately with more HybridomaSelection Medium, based on a final plating of 5×10⁶ B cells total per96-well plate and 200 μl per well. The cells were mixed gently andpipetted into 96-well plates and allowed to grow. On day 7 or 10, halfof the medium was removed, and the cells were re-fed with HybridomaSelection Medium.

Example 5 Selection of Candidate Antibodies by ELISA

After 14 days of culture, primary screening of hybridoma supernatantsfrom the cohort #1 (mice in cohort one were split arbitrarily intofusion #1 and #2) for HER-3-specific antibodies was performed by ELISAusing purified his-tagged HER-3 ECD and counter-screening against anirrelevant his-tagged protein by ELISA using goat anti-huIgGFc-HRP(Caltag Inc., Cat. No. H10507, using concentration was 1:2000 dilution)to detect human IgG binding to HER-3 ECD immobilized on ELISA plates.The old culture supernatants from positive hybridoma cells growth wellsbased on primary screen were removed, and the HER-3 positive hybridomacells were suspended with fresh hybridoma culture medium and weretransferred to 24-well plates. After 2 days in culture, thesesupernatants were used for a secondary confirmation screen. In thesecondary confirmation screen for HER-3 specific fully human IgGkantibodies, the positives in the first screening were screened by ELISAwith two sets of detective antibodies: goat anti-huIgGFc-HRP (CaltagInc., Cat. No. H10507, using a 1:2000 dilution) for human gamma chaindetection, and goat anti-hIg kappa-HRP (Southern Biotechnology, Cat. No.2060-05) for human kappa light chain detection. From cohort #1, 91 fullyhuman IgG/kappa HER-3 specific monoclonal antibodies were generated.

Example 6 Selection of Candidate Antibodies by FMAT/FACS

After 14 days of culture, hybridoma supernatants from the cohorts #3 and#4 (fusions #3 and #4) were screened for HER-3-specific monoclonalantibodies by FMAT. In the primary screen, hybridoma supernatants at1:10 final dilution were incubated with Rat1-HER-3 cells expressinghuman HER-3 and 400 ng/ml Cy5-conjugated Goat F(ab′)2 anti-human IgG,Fc-specific antibody (Jackson ImmunoResearch, Cat. No. 109-176-098) atroom temperature for 6 hours. The binding of antibodies and detectionantibodies to cells were measured by FMAT (Applied Biosystems).Non-specific binding of antibodies to the cells was determined by theirbinding to parental Rat1 cells. A total of 420 hybridomas producingHER-3-specific antibodies were selected from the primary screen offusion #3. The supernatants from these expanded cultures were testedagain using the same FMAT protocol, and 262 of them were confirmed tobind specifically to HER-3 expressing cells. A total of 193 hybridomasproducing HER-3 specific antibodies were selected from the primaryscreen of fusion #4. The supernatants from these expanded cultures weretested by FACS, and 138 of them were confirmed to bind specifically tocells expressing HER-3. In the FACS confirmation assay, Rat1-XHER-3cells and parental Rat1 cells (as negative control) were incubated withhybridoma supernatants at 1:2 dilution for 1 hour at 40° C. in PBScontaining 2% FBS. Following washing with PBS, the binding of antibodiesto the cells was detected by 2.5 μg/ml Cy5-conjugated Goat F(ab′)2anti-human IgG, Fc-specific antibody (JIR #109-176-098) and 5 μg/mlPE-conjugated Goat F(ab′)2 anti-human kappa-specific antibody (SB#2063-09). After removing unbound antibodies by washing with PBS, thecells were fixed by cytofix (BD #51-2090KZ) at 1:4 dilution and analyzedby FACSCalibur.

Example 7 Selection of Hybridomas for Cloning

Antibodies from cohort #1 were selected for hybridoma cloning based onspecificity for HER-3 over HER1 (EGF-R), HER-2 and HER-4 in ELISA usingpurified recombinant extra-cellular domains (available from, forexample, R&D Biosystems, Minneapolis, Minn.), FACS-based analysis ofhuman tumor cell lines expressing different HER family members, anda >5-time increase in mean fluorescent intensity in FACS staining forHER-3 positive cells over background. Based on these criteria, a totalof 23 hybridoma lines were selected for cloning by limiting dilutioncell plating.

Antibodies from cohorts 3 and 4 were selected for hybridoma cloningbased on specificity for HER-3 over HER-1 (EGF-R), HER-2 and HER-4 plusthree other criteria. The first criterion was an ELISA screen forantibodies with epitopes contained within the L2 domain of HER-3 (see,Example 8 below).

The second criterion was neutralization of binding of biotinylatedheregulin-alpha to HER-3 expressing cells in a FACS based assay. SKBR-3cells were harvested, washed in culture medium, pelleted viacentrifugation and resuspended in culture medium. Resuspended cells werealiquoted into 96-well plates. The plates were centrifuged to pellet thecells. Test antibodies in exhaust hybridoma supernatants were added at25 μl/well and incubated for 1 hour on ice to allow antibody binding.Fifty μl of a 10 nM heregulin-alpha (R&D Biosystems) solution was addedto each well for a final concentration of 5 nM and incubated on ice for1.5 hours. Cells were washed in 150 μl PBS, pelleted by centrifugationand the supernatant removed. Cells were resuspended in 50 μl of goatanti-HRG-alpha polyclonal antibody at 10 μg/ml and incubated for 45minutes on ice. Cells were washed in 200 μl PBS, pelleted bycentrifugation, and the supernatant was removed. Fifty μl of a solutionof rabbit Cy5-labeled anti-goat polyclonal antibody at 5 μg/ml plus 7AADat 10 μg/ml was added and incubated on ice for 15 minutes. Cells werewashed in 200 μl PBS, pelleted by centrifugation and the supernatantremoved. The cells were resuspended in 100 μl of FACS buffer and read inthe FACS. Test HER-3 antibodies that reduced binding of heregulin-alphawere those that had lowest fluorescence intensity. As positive controls,1:5 serial dilutions from 10,000 ng/ml to 16 ng/ml of a mouse HER-3 mAb(105.5) or the human IgG1 HER-3 mAb, U1-49 was used. Negative controlswere heregulin-alpha alone, cells alone, goat anti-heregulin-alphapolyclonal antibody alone and rabbit Cy5-labeled anti-goat polyclonalantibody alone.

The third criterion was relative ranking for affinity and/or higherrelative mean fluorescence intensity in FACS using HER-3 expressing celllines. Relative ranking for affinity was performed by normalizingHER-3-specific antibody concentrations and plotting versus data fromlimiting antigen ELISA as follows.

Normalization of antigen specific antibody concentrations using highantigen ELISA: Using an ELISA method, supernatants for concentration ofantigen specific antibody were normalized. Using two anti-HER-3 humanIgG1 antibodies from cohort 1 of known concentration titrated inparallel, a standard curve was generated and the amount of antigenspecific antibody in the test hybridoma supernatants from cohorts 3 and4 were compared to the standard. In this way, the concentration of humanHER-3 IgG antibody in each hybridoma culture was estimated.

Neutravidin plates were made by coating neutravidin at 8 μg/ml in 1×PBS/0.05% sodium azide on Costar 3368 medium binding plates at 50μl/well with overnight incubation at 4° C. The next day the plates wereblocked with 1× PBS/1% skim milk. Photobiotinylated his-tagged-HER-3 ECDat 500 ng/ml in 1× PBS/1% skim milk was bound to the neutravidin platesby incubating for 1 hour at room temperature. Hybridoma supernatant,serially diluted 1:2.5 from a starting dilution of 1:31 to a finaldilution of 1:7568 in 1× PBS/1% skim milk/0.05% azide, was added at 50μl/well, and then incubated for 20 hours at room temperature. Serialdilutions were used to ensure obtaining OD readings for each unknown inthe linear range of the assay. Next, a secondary detection antibody,goat anti human IgG Fc HRP at 400 ng/ml in 1× PBX/1% skim milk was addedat 50 μl/well. After 1 hour at room temperature, the plates were againwashed 5 times with water and 50 μl of one-component TMB substrate wereadded to each well. The reaction was stopped after 30 minutes byaddition of 50 μl 1M hydrochloric acid to each well and the plates wereread at wavelength 450 nm. A standard curve was generated from the twoIgG1 HER-3 mAbs from cohort #1, serially diluted at 1:2 from 1000 ng/mlto 0.06 ng/ml and assessed in ELISA using the above protocol. For eachunknown, OD readings in the linear range of the assay were used toestimate the concentration of human HER-3 IgG in each sample.

The limited antigen analysis is a method that affinity ranks theantigen-specific antibodies prepared in B-cell culture supernatantsrelative to all other antigen-specific antibodies. In the presence of avery low coating of antigen, only the highest affinity antibodies shouldbe able to bind to any detectable level at equilibrium. (See, e.g., PCTPublication No. WO 03048730A2). In this instance, two mAbs from cohort#1, both of known concentration and known KD, were used as benchmarks inthe assay.

Neutravidin plates were made by coating neutravidin at 8 μg/ml in 1×PBS/0.05% sodium azide on Costar 3368 medium binding plates at 50μl/well with overnight incubation at 4° C. The next day the plates wereblocked with 1× PBS/1% skim milk. Biotinylated his-tagged-HER-3 ECD (50μl/well) was bound to 96-well neutravidin plates at five concentrations:125, 62.5, 31.2, 15.6, and 7.8 ng/ml in 1× PBS/1% skim milk for 1 hourat room temperature. Each plate was washed 5 times with water. Hybridomasupernatants diluted 1:31 in 1× PBS/1% skim milk/0.05% azide were addedat 50 ul/well. After 20 hours incubation at room temperature on ashaker, the plates were again washed 5 times with dH₂O. Next, asecondary detection antibody, goat anti human IgG Fc HRP (Horse RadishPeroxidase) at 400 ng/ml in 1× PBS/1% skim milk was added at 50 μl/well.After 1 hour at room temperature, the plates were again washed 5 timeswith dH₂O and 50 μL of one-component TMB substrate were added to eachwell. The reaction was stopped after 30 minutes by addition of 50 μl of1M hydrochloric acid to each well and the plates were read at wavelength450 nm. OD readings from an antigen concentration that yielded OD valuesin the linear range were used in for data analysis.

Plotting the high antigen data (which comparatively estimate specificantibody concentrations; see above for details) versus the limitedantigen OD illustrated that the relatively higher affinity antibodies,e.g., those that bound had higher OD in the limited antigen assay whilehaving lower amounts of IgG HER-3 antibody in the supernatant.Hybridomas from cohorts #3 and #4 for the 33 best performing antibodiesin these sets of assays were advanced to cloning by limiting dilutionhybridoma plating.

Alternatively, FACS analysis of HER-3 expression of RatI/pLXSN andRatI/HER-3 cells showed similar results (no crossreactivity withendogenous rat epitopes. In detail, 1×10⁵ cells were harvested with 10mM EDTA in PBS, washed once with FACS buffer (PBS, 3% FCS, 0.4% azide)and seeded on a 96-well round bottom plate. The cells were spun for 3minutes at 1000 rpm to remove supernatant and then resuspended with thespecific HER-family antibodies (3 μg/ml). Cell suspensions wereincubated on ice for 45 minutes, washed twice with FACS buffer andresuspended with secondary antibody (100 μl/well) donkey-anti-human-PE(Jackson Immunoresearch, PA) diluted 1:50 in FACS buffer. The cellsuspensions were incubated on ice and in the dark for 30 minutes, washedtwice with FACS buffer and analyzed (FACS, Beckman Coulter).

Example 8 Structural Analysis of Anti-HER-3 Antibodies

The following discussion provides structural information related toantibodies prepared as described herein. In order to analyze structuresof the antibodies, genes encoding the heavy and light chain fragmentswere amplified out of the particular hybridoma. Sequencing wasaccomplished as follows:

The V_(H) and V_(L) transcripts were amplified from individual hybridomaclones in 96 well plate using reverse transcriptase polymerase chainreaction (RT-PCR). Poly(A)+-mRNA was isolated from approximately 2×10⁵hybridoma cells using a Fast-Track kit (Invitrogen). Four PCR reactionswere run for each Hybridoma: two for light chain (kappa (κ), and two forgamma heavy chain (γ). The QIAGEN OneStep room temperature-PCR kit wasused for amplification (Qiagen, Catalog No. 210212). In the coupled roomtemperature-PCR reactions, cDNAs were synthesized with blend of roomtemperature enzymes (Omniscript and Sensiscript) using antisensesequence specific primer corresponded to C-κ, or to a consensus of theCH1 regions of Cγ genes. Reverse transcription was performed at 50° C.for 1 hr followed by PCR amplification of the cDNA by HotStarTaq DNAPolymerase for high specificity and sensitivity. Each PCR reaction useda mixture of 5′-sense primers; primer sequences were based on the leadersequences of V_(H) and V_(K) available at the Vbase website(http://vbase.mrc-cpe.cam.ac.uk/).

PCR reactions were run at 94° C. for 15 min, initial hot start followedby 40 cycles of 94° C. for 30 sec (denaturation), 60° C. for 30 sec(annealing) and 72° C. for 1 min (elongation).

PCR products were purified and directly sequenced using forward andreverse PCR primers using the ABI PRISM BigDye terminator cyclesequencing ready reaction Kit (Perkin Elmer). Both strands weresequenced using Prism dye-terminator sequencing kits and an ABI 377sequencing machine.

Sequence analysis: Analyses of human V heavy and V kappa cDNA sequencesof the HER-3 antibodies were accomplished by aligning the HER-3sequences with human germline V heavy and V kappa sequences usingAbgenix in-house software (5AS). The software identified the usage ofthe V gene, the D gene and the J gene as well as nucleotide insertionsat the recombination junctions and somatic mutations. Amino acidsequences were also generated in silico to identify somatic mutations.Similar results could be obtained with commercially available sequenceanalysis software and publicly available information on the sequence ofhuman V, D, and J genes, e.g., Vbase (http://vbase.mrc-cpe.cam.ac.uk/).

Molecular cloning of mAb U1-59: Total RNA was extracted from the tissueculture well containing multiple hybridomas lineages, including thehybridoma lineage secreting antibody U1-59. A heavy chain variableregion was amplified using 5′-leader VH family specific primers, with3′-C-gamma primer. A major band was amplified using a VH4 primer, noother bands were visible. The VH4-34 gamma fragment was cloned intopCDNA expression vector in frame with a human gamma 1 constant regiongene.

An IgM heavy chain variable region was amplified using 5′ VH familyspecific primers with 3′ mu constant region primer. A major band wasamplified using VH2 primer, no other bands were visible. The VH2-5 mufragment was cloned into pCDNA expression vector in frame with a humanmu constant region gene. V kappa chains were amplified and sequenced.Four kappa chain RT-PCR products were identified. The products weresequenced and after sequence analysis via in silico translation, onlythree of them had open-reading frames. These three functional kappachains were cloned out of the oligoclonal U1-59 hybridoma wellidentified based on V kappa gene usage as (1) VK1 A3-JK2, (2) VK1A20-JK3 and (3) B3-JK1. All V-kappa were cloned into pCDNA expressionvector in frame with a human kappa light chain constant region gene.

Transfections: Each heavy chain was transfected with each of the kappachains in transient transfections for a total of 6 heavy chain/kappalight chain pairs. The transfection of the gamma chain with the A20kappa chain gave poor antibody expression, while no antibody wassecreted or detected when the A20 kappa chain was co-transfected withthe mu chain. A total of three IgG sups and two IgM sups were availablefor HER-3 binding assay.

Chain VH D J Constant ORF Heavy VH4-34 D1-20 JH2 Gamma Yes Heavy VH2-5D6-6 JH4b Mu Yes Light A3 JK2 Kappa Yes Light A20 JK3 Kappa Yes Light B3JK1 Kappa Yes Light A27 JK3 Kappa NO

Binding activity to HER-3+ cell lines was detected in FACS with the IgG1mAb consisting of the VH4-34 and the B3 kappa chain. No other VH/Vkcombinations gave fluorescence signal above background in FACS usingHER-3+ cell lines.

Binding competition of the anti-HER-3 antibodies: Multiplexedcompetitive antibody binning was performed as published in Jia et al.(2004) J Immunol Methods. 288, 91-98 to assess clusters of HER-3antibodies that competed for binding to HER-3. Tested HER-3 antibodiesfrom cohort 1 clustered into 5 bins based on competition for binding.

Bin#1 Bin#2 Bin#3 Bin#4 Bin#5 U1-42 U1-48 U1-52 U1-38 U1-45 U1-44 U1-50U1-39 U1-40 U1-62 U1-51 U1-41 U1-46 U1-43 U1-47 U1-49 U1-61 U1-58 U1-53U1-55

Epitope characterization of anti-HER-3 antibodies: The epitopes of humananti-HER-3 antibodies were characterized. First a dot blot analysis ofthe reduced, denatured HER-3-His tagged purified ECD protein showedabsence of binding by the anti-HER-3 antibodies tested (U1-59, U1-61,U1-41, U1-46, U1-53, U1-43, U1-44, U1-47, U1-52, U1-40, U1-49))demonstrating that all had epitopes sensitive to reduction of disulfidebonds, suggesting that all had discontinuous epitopes. Next, theantibodies were mapped to defined domains in the HER-3 molecule byengineering various human-rat HER-3 chimeric molecules, based on thedivision of the HER-3 extra-cellular domain into four domains:

-   -   1) L1 (D1): the minor ligand-binding domain,    -   2) S1 (D2): the first cysteine-rich domain,    -   3) L2 (D3): the major ligand-binding domain, and    -   4) S2 (D4): the sec cysteine-rich domain.

The extra-cellular domain (ECD) of Human HER-3 cDNA was amplified fromRAT1-HER-3 cells. The rat HER-3 cDNAs was amplified by RT-PCR from ratliver RNA and confirmed by sequencing. The cDNAs expressing the ECD ofhuman and rat HER-3 were cloned into mammalian expression vectors asV5-His fusion proteins. Domains from the human HER-3 ECD were swappedinto the scaffold provided by the rat HER-3 ECD by using the Mfe1, BstX1and DraIII internal restriction sites. By this means, various chimericrat/human HER-3 ECD HIS fusion proteins (amino acids 1-160, 161-358,359-575, 1-358, 359-604) were constructed and expressed via transienttransfection of HEK 293T cells. Expression of the constructs wasconfirmed using a rat polyclonal antibody against human HER-3. The humanmonoclonal antibodies were tested in ELISA for binding to the secretedchimeric ECDs.

Two of the human antibodies, including antibody U1-59, cross-reactedwith rat HER-3. To assign binding domains, these mAbs were testedagainst a truncated form of HER-3 consisting of L1-S1-V5his taggedprotein purified from the supernatant of HEK 293T cells transfected witha plasmid DNA encoding the expression of the L1-S1 extra-cellulardomains of HER-3. mAb U1-59 bound to the L1-S1 protein in ELISA,implying that its epitope is in L1-S1. mAb 2.5.1 did not bind to theL1-S1 protein, implying that its epitope is in L2-S2. Further mapping ofantibody U1-59 was accomplished using SELDI time of flight massspectroscopy with on-chip proteolytic digests of mAb-HER-3 ECDcomplexes.

Mapping U1-59 epitopes using SELDI: Further mapping of antibody U1-59was accomplished using a SELDI time of flight mass spectroscopy withon-chip proteolytic digests of mAb-HER-3 ECD complexes. Protein A wascovalently bound to a PS20 protein chip array and used to capture mAbU1-59. Then the complex of the PS20 protein chip and the monoclonalantibody was incubated with HER-3-His purified antigen. Next theantibody-antigen complex was digested with high concentration of Asp-N.The chip was washed, resulting in retention of only the HER-3 peptidebound to the antibody on the chip. The epitope was determined by SELDIand identified by mass of the fragment. The identified 6814 D fragmentcorresponds to two possible expected peptides generated from a partialdigest of the HER-3-his ECD. Both overlapping peptides map to the domainS1. By coupling SELDI results with binding to a HER-3 deletionconstruct, the epitope was mapped to residues 251 to 325.

The location of the binding domains in the extracellular part of HER-3that are recognized by the human anti-HER-3 mAbs are summarized in TABLE4. The epitope domain mapping results were consistent with results fromantibody competition binding competition bins, with antibodies thatcross-competed each other for binding to HER-3 also mapping to the samedomains on HER-3.

TABLE 4 A summary of mAb binding domains based on ELISA assay resultsBinding Binding MAb Rat XR domain mAb Rat XR domain U1-59 Yes S1 U1-2 NoL2 U1-61 No L2 U1-7 No L2 U1-41 No L2 U1-9 No L2 U1-46 No S1 U1-10 No L2U1-53 No L2 U1-12 No L2 U1-43 No L2 U1-13 No L2 U1-44 No S1 U1-14 No L2U1-47 No S1 U1-15 No L2 U1-52 Yes L2S2 U1-19 No L2 U1-40 No L2 U1-20 NoL2 U1-49 No L1 U1-21 No L2 U1-21 No L2 U1-28 No L2 U1-22 No L2 (U1-31)No L2 U1-23 No L2 U1-32 No L2 U1-24 No L2 (U1-35) No L2 U1-25 No L2U1-36 No L2 U1-26 No L2 (U1-37) No L2 U1-27 No L2 XR = cross-reactive

Example 9 Determination of Canonical Classes of Antibodies

Antibody structure has been described in terms of “canonical classes”for the hypervariable regions of each immunoglobulin chain (Chothia etal. (1987) J. Mol. Biol. 196:901-17). The atomic structures of the Faband VL fragments of a variety of immunoglobulins were analyzed todetermine the relationship between their amino acid sequences and thethree-dimensional structures of their antigen binding sites. Chothia, etal. found that there were relatively few residues that, through theirpacking, hydrogen bonding or the ability to assume unusual phi, psi oromega conformations, were primarily responsible for the main-chainconformations of the hypervariable regions. These residues were found tooccur at sites within the hypervariable regions and in the conservedβ-sheet framework. By examining sequences of immunoglobulins havingunknown structure, Chothia, et al. show that many immunoglobulins havehypervariable regions that are similar in size to one of the knownstructures and additionally contained identical residues at the sitesresponsible for the observed conformation.

Their discovery implied that these hypervariable regions haveconformations close to those in the known structures. For five of thehypervariable regions, the repertoire of conformations appeared to belimited to a relatively small number of discrete structural classes.These commonly occurring main-chain conformations of the hypervariableregions were termed “canonical structures.” Further work by Chothia etal. (Nature (1989) 342:877-83) and others (Martin et al. (1996) J. Mol.Biol. 263:800-15) confirmed that there is a small repertoire ofmain-chain conformations for at least five of the six hypervariableregions of antibodies.

The CDRs of each antibody described above were analyzed to determinetheir canonical class. As is known, canonical classes have only beenassigned for CDR1 and CDR2 of the antibody heavy chain, along with CDR1,CDR2 and CDR3 of the antibody light chain. The tables below summarizethe results of the analysis. The canonical class data is in the form ofHCDR1-HCDR2-LCDR1-LCDR2-LCDR3, wherein “HCDR” refers to the heavy chainCDR and “LCDR” refers to the light chain CDR. Thus, for example, acanonical class of 1-3-2-1-5 refers to an antibody that has a HCDR1 thatfalls into canonical class 1, a HCDR2 that falls into canonical class 3,a LCDR1 that falls into canonical class 2, a LCDR2 that falls intocanonical class 1, and a LCDR3 that falls into canonical class 5.

Assignments were made to a particular canonical class where there was70% or greater identity of the amino acids in the antibody with theamino acids defined for each canonical class. The amino acids definedfor each antibody can be found, for example, in the articles by Chothia,et al. referred to above. TABLE 5 and TABLE 6 report the canonical classdata for each of the HER-3 antibodies. Where there was less than 70%identity, the canonical class assignment is marked with an asterisk(“*”) to indicate that the best estimate of the proper canonical classwas made, based on the length of each CDR and the totality of the data.Where there was no matching canonical class with the same CDR length,the canonical class assignment is marked with a letter s and a number,such as “s18”, meaning the CDR is of size 18. Where there was nosequence data available for one of the heavy or light chains, thecanonical class is marked with “Z”.

TABLE 5 Antibody (sorted) H1-H2-L1-L2-L3 H3length U1-38 3-1-4-1-1 9U1-39 1-1-4-1*-1 6 U1-40 3-1-4-1-1 15 U1-41 3-1-2-1-1 15 U1-42 1-2-2-1-19 U1-43 3-1-2-1-1 17 U1-44 1-2-2-1-1 9 U1-45 1-2*-2-1-1 16 U1-463-s18-Z-Z-Z 17 U1-47 3-s18-2-1-1 16 U1-48 1-1-Z-Z-Z 16 U1-49 1-3-4-1-117 U1-50 3-1-2-1-1 17 U1-51 1-1-3-1-1 19 U1-52 3-1-8-1-1 15 U1-531-3-2-1-1 10 U1-55 3-1-4-1-1 15 U1-57 3-1-4-1-1 15 U1-58 1-3-2-1-1 12U1-59 1-1-3-1-1 9 U1-61.1 3-1*-2-1-1 16 U1-62 1-2-8-1-1 12 U1-23-1-2-1-1 12 U1-7 3-1-2-1-1 12 U1-9 3-1-2-1-1 12 U1-10 3-1-2-1-1 12U1-12 3-1-2-1-1 12 U1-13 3-1-4-1-1 7 U1-14 3-1-2-1-1 12 U1-15 3-1-8-1-114 U1-19 3-1-Z-Z-Z 12 U1-20 3-1-2-1-1 19 U1-21 3-1-2-1-1 12 U1-223-1-2-1-1 12 U1-23 3-1-2-1-1 12 U1-24 3-1-2-1-1 12 U1-25 3-1-2-1-1 12U1-26 3-1-2-1-1 12 U1-27 3-1-2-1-1 12 U1-28 3-1-2-1-1 12 U1-31 1-2-2-1-113 U1-32 3-1-2-1-1 12 U1-35 1-3-2-1-1 14 U1-36 3-1-2-1-1 12 U1-371-2-Z-Z-Z 13

TABLE 6 is an analysis of the number of antibodies per class. The numberof antibodies having the particular canonical class designated in theleft column is shown in the right column. The four mAbs lacking onechain sequence data and thus having “Z” in the canonical assignment arenot included in this counting.

The most commonly seen structure is 3-1-2-1-1: Twenty-one out offorty-one mAbs having both heavy and light chain sequences had thiscombination.

TABLE 6 H1-H2-L1-L2-L3 Count 1-1-3-1-1 2 1-1-4-1*-1 1 1-2-2-1-1 41-2-8-1-1 1 1-3-2-1-1 3 1-3-4-1-1 1 3-1-2-1-1 21 3-1-4-1-1 5 3-1-8-1-1 23-s18-2-1-1 1

Example 10 Determination of Antibody Affinity

Affinity measurements of anti-HER-3 antibodies were performed byindirect FACS Scatchard analysis. Therefore, 10⁵ cells of interest orSK-Br 3 cells were harvested with 10 mM EDTA in PBS, washed once withFACS buffer (PBS, 3% FCS, 0.4% azide) and seeded on a 96-well roundbottom plate. The cells were spun for 3 min at 1000 rpm to removesupernatant and then resuspended with α-HER-3 antibody (3 μg/ml) or withantibody dilutions (100 μl/well) starting with 20 μg/ml human monoclonalantibody in FACS buffer, diluted in 1:2 dilution steps. Cell suspensionswere incubated on ice for 1 hr, washed twice with FACS buffer andresuspended with secondary antibody (100 μl/well) donkey-anti-human-PE(Jackson) diluted 1:50 in FACS buffer. The cell suspensions wereincubated on ice and in the dark for 30 min, washed twice with FACSbuffer and analyzed (FACS, Beckman Coulter). According to the FACSScatchard analysis, the fluorescence mean was calculated for eachmeasurement. Background staining (=without 1^(st) antibody) wassubtracted from each fluorescence mean. Scatchard plot withx-value=fluorescence mean and y-value=fluorescence mean/concentration ofmAb (nM) was generated. The KD was taken as the absolute value of 1/m oflinear equation. Affinity measurements for certain antibodies selectedin this manner are provided in TABLE 7.

TABLE 7 Clone KD (nm) U1-38 n.d. U1-39 102 U1-40 6.7 U1-41 0.18 U1-42n.d. U1-43 0.57 U1-44 4 U1-52 16.8 U1-61 0.13 U1-62 20.4 U1-46 13.8U1-47 9.38 U1-49 1 U1-50 39.3 U1-51 131.6 U1-53 0.082 U1-55.1 3.7 U1-586.4 U1-59 3.69 U1-24 0.06 U1-7 0.02

Example 11 Anti-HER-3 Antibodies Induce HER-3 Receptor Endocytosis

HER-3 has been identified as a factor that can influence initiation andprogression of hyperproliferative diseases through serving as animportant gatekeeper of HER family mediated cell signaling. Thus, ifHER-3 is effectively cleared from the cell surface/membrane by receptorinternalization, cell signaling and therefore transformation and/ormaintenance of cells in malignancy can be ultimately diminished orsuppressed.

In order to investigate whether anti-HER-3 antibodies are capable ofinducing accelerated endocytosis of HER-3, the relative amount of HER-3molecules on the cell surface after 0.5 and 4 hr incubation of the cellswith anti-HER-3 antibodies were compared. 3×10⁵ cells were seeded innormal growth medium in 24-well dish and left to grow overnight. Cellswere preincubated with 10 μg/ml anti-HER-3 mAbs in normal growth mediumfor the indicated times at 37° C. Cells were detached with 10 mM EDTAand incubated with 10 μg/ml anti-HER-3 mAbs in wash buffer (PBS, 3% FCS,0.04% azide) for 45 min at 4° C. Cells were washed twice with washbuffer, incubated with donkey-anti-human-PE secondary antibody (Jackson)diluted 1:100 for 45 min at 4° C., washed twice with wash buffer andanalyzed by FACS (BeckmanCoulter, EXPO). Percent internalization wascalculated based on the reduction of the mean fluorescence intensity ofanti-HER-3 treated samples relative to control-treated samples. Theseexperiments demonstrated that treatment of cells with anti-HER-3antibodies led to internalization of the receptor. See, FIG. 5 of USPublication No. 20080124345.

Example 12 Inhibition of Ligand Binding to Human Cancer Cells SKBr3 byHuman Anti-HER-3 Antibodies

Radioligand competition experiments were performed in order toquantitate the ability of the anti-HER-3 antibodies to inhibit ligandbinding to HER-3 in a cell based assay. Therefore, the HER-3 receptorbinding assay was performed with 4×10⁵ SK-BR-3 cells which wereincubated with varying concentrations of antibodies for 30 min on ice.1.25 nM [I¹²⁵]-α-HRG/[¹²⁵I]-β-HRG were added to each well and theincubation was continued for 2 hr on ice. The plates were washed fivetimes, air-dried and counted in a scintillation counter. The antibodieswere capable of specifically reducing the binding of[¹²⁵I]-α-HRG/[¹²⁵I]-β-HRG to cells expressing endogenous HER-3. See,FIGS. 6a-6e of US Publication No. 20080124345.

Example 13 Inhibition of Ligand-Induced HER-3 Phosphorylation by HumanAnti-HER-3 Antibodies

ELISA experiments were performed in order to investigate whether theantibodies are able to block ligand β-HRG-mediated activation of HER-3.Ligand-mediated HER-3 activation was detected by increased receptortyrosine phosphorylation.

Day 1: 1×96 well dish was coated with 20 μg/ml Collagen I in 0.1 Macetic acid for 4 hr at 37° C. 2.5×10⁵ cells were seeded in normalgrowth medium

Day 2: Cells were starved in 100 μl serum free medium for 24 hr.

Day 3: Cells were preincubated with 10 μg/ml anti-HER-3 mAbs for 1 hr at37° C. and then treated with 30 ng/ml β-HRG-EGF domain (R&D Systems) for10 min. Medium was flicked out and cells were fixed with 4% formaldehydesolution in PBS for 1 hr at room temperature. Formaldehyde solution wasremoved and cells were washed with wash buffer (PBS/0.1% Tween 20).Cells were quenched with 1% H₂O₂, 0.1% NaN₃ in wash buffer and incubatedfor 20 min at room temperature, then blocked with NET-Gelantine for 5 hrat 4° C. Primary antibody phospho-HER-3 (Tyr1289) (polyclonal rabbit;Cell signaling #4791; 1:300) was added overnight at 4° C.

Day 4: The plate was washed 3× with wash buffer, then incubated withanti-rabbit-POD diluted 1:3000 in PBS−0.5% BSA was added to each welland incubated for 1.5 hr at room temperature. The plate was washed 3×with wash buffer and once with PBS. Tetramethylbenzidine (TMB,Calbiochem) was added and monitored at 650 nm. The reaction was stoppedby addition of 100 μl 250 nM HCl and the absorbance was read at 450 nmwith a reference wavelength of 650 nm using a Vmax plate reader (ThermoLab Systems).

These experiments demonstrated that anti-HER-3 antibodies were able toreduce ligand-mediated HER-3 activation as indicated by decreasedreceptor tyrosine phosphorylation. See, FIG. 7a of US Publication No.20080124345.

To test potency of mAb U1-53 to inhibit ligand induced HER-3 activation,MCF-7 cells were starved for 24 hr, incubated with mAb U1-53 for 1 hr at37° C. and stimulated with 10 nM HRG-β for 10 min. Lysates weretransferred to 1B4 (mouse anti-HER-3 mAb) ELISA plates andphosphorylation of HER-3 was analyzed with antibody 4G10.Phosphorylation of HER-3 was almost completely inhibited in a dosedependent manner with an IC₅₀ of 0.14 nM. See, FIG. 7b of US PublicationNo. 20080124345.

Example 14 Inhibition of Ligand-Induced p42/p44 MAP-KinasePhosphorylation by Human Anti-HER-3 Antibodies

Next ELISA experiments were performed in order to investigate whetherthe antibodies are able to block ligand β-HRG-mediated activation ofp42/p44 MAP-Kinase. Ligand-mediated HER-3 activation was detected byincreased protein (Thr202/Tyr204) phosphorylation.

Day 1: 1×96 well dish was coated with 20 μg/ml Collagen I in 0.1 Macetic acid for 4 hr at 37° C. 3×10⁵ cells were seeded in normal growthmedium

Day 2: Cells were starved in 100 μl serum free medium for 24 hr.

Day 3: Cells were preincubated with 5 μg/ml anti-HER-3 mAbs for 1 hr at37° C. and then treated with 20 ng/ml β-HRG-EGF domain (R&D Systems) for10 min. Medium was flicked out and cells were fixed with 4% formaldehydesolution in PBS for 1 hr at room temperature. Formaldehyde solution wasremoved and cells were washed with wash buffer (PBS/0.1% Tween 20).Cells were quenched with 1% H₂O₂, 0.1% NaN₃ in wash buffer and incubatedfor 20 min at room temperature, then blocked with PBS/0.5% BSA for 5 hrat 4° C. Primary antibody phospho-p44/p42 MAP Kinase (Thr202/Tyr204)(polyclonal rabbit; Cell signaling #9101; 1:3000) was added overnight at4° C.

Day 5: The plate was washed 3× with wash buffer, then incubated withanti-rabbit-HRP diluted 1:5000 in PBS−0.5% BSA was added to each welland incubated for 1.5 hr at room temperature. The plate was washed 3×with wash buffer and once with PBS. Tetramethylbenzidine (TMB,Calbiochem) was added and monitored at 650 nm. The reaction was stoppedby addition of 100 μl 250 nM HCl and the absorbance was read at 450 nmwith a reference wavelength of 650 nm using a Vmax plate reader (ThermoLab Systems). These experiments revealed that the antibodies were ableto reduce ligand-mediated p42/p44 MAP-Kinase activation as indicated bydecreased phosphorylation. See, FIG. 8 of US Publication No.20080124345.

Example 15 Inhibition of β-HRG-Induced Phospho-AKT Phosphorylation byHuman Anti-HER-3 Antibodies

In the following ELISA experiment we investigated whether the anti-HER-3antibodies are able to block ligand β-HRG-mediated activation ofAKT-Kinase. Ligand-mediated AKT activation was detected by increasedprotein (Ser473) phosphorylation.

Day 1: 1×96 well dish was coated with 20 μg/ml Collagen I in 0.1 Macetic acid for 4 hr at 37° C. 3×10⁵ cells were seeded in normal growthmedium

Day 2: Cells were starved in 100 μl serum free medium for 24 hr.

Day 3: Cells were preincubated with 5 μg/ml anti-HER-3 mAbs for 1 hr at37° C. and then treated with 20 ng/ml β-HRG-EGF domain (R&D Systems) for10 min. Medium was flicked out and cells were fixed with 4% formaldehydesolution in PBS for 1 hr at room temperature. Formaldehyde solution wasremoved and cells were washed with wash buffer (PBS/0.1% Tween 20).Cells were quenched with 1% H₂O₂, 0.1% NaN₃ in wash buffer and incubatedfor 20 min at room temperature, then blocked with PBS/0.5% BSA for 5 hrat 4° C. Primary antibody phospho-Akt (Ser473) (polyclonal rabbit; Cellsignaling #9217; 1:1000) was added overnight at 4° C.

Day 4: The plate was washed 3× with wash buffer, then incubated withanti-rabbit-HRP diluted 1:5000 in PBS−0.5% BSA was added to each welland incubated for 1.5 hr at room temperature. The plate was washed 3×with wash buffer and once with PBS. Tetramethylbenzidine (TMB,Calbiochem) was added and monitored at 650 nm. The reaction was stoppedby addition of 100 μl 250 nM HCl and the absorbance was read at 450 nmwith a reference wavelength of 650 nm using a Vmax plate reader (ThermoLab Systems). The anti-HER-3 antibodies were able to reduceβ-HRG-mediated AKT as indicated by decreased phosphorylation. See, FIG.9 of US Publication No. 20080124345.

Example 16 Inhibition of α-HRG/β-HRG-Mediated MCF7 Cell Proliferation byHuman Anti-HER-3 Antibodies

In vitro experiments were conducted in order to determine the ability ofthe antibodies to inhibit HRG-stimulated cell proliferation. 2000 MCF7cells were seeded in FCS-containing medium on 96-well plates overnight.Cells were preincubated in quadruplicates with antibody diluted inmedium with 0.5% FCS for 1 hr at 37° C. Cells were stimulated with 30ng/ml α- or 20 ng/ml β-HRG (R&D Systems) by adding ligand directly toantibody solution and were then left to grow for 72 hr. ALAMAREBLUE™(BIOSOURCE) was added and incubated at 37° C. in the dark. Absorbancewas measured at 590 nm every 30 min. The data were taken 90 min afteraddition of alamar blue. These studies showed that representativeantibodies could inhibit HRG-induced cell growth in human cancer cells.See, FIG. 10 of US Publication No. 20080124345.

Example 17 Inhibition of β-HRG-Induced MCF7 Cell Migration by HumanAnti-HER-3 Antibodies

Transmigration experiments were performed in order to investigatewhether the antibodies block cell migration. Serum-starved MCF7 cellswere preincubated by adding the indicated amount of antibody to the cellsuspension and incubating both for 45 min at 37° C. 500 μl cellsuspension (50,000 cells) was then placed in the top chamber of collagenI-coated transwells (BD Falcon, 8 μm pores). 750 μl medium (MEM, aminoacids, Na-pyruvate, Pen.-Strept., 0.1% BSA, without fetal calf serum)alone or containing the ligands β-HRG-EGF domain (R&D Systems) were usedin the bottom chamber. Cells were left to migrate for 8 hr at 37° C. andwere stained with DAPI. Stained nuclei were counted manually; percentinhibition was expressed as inhibition relative to a control antibody.These experiments demonstrated that representative anti-HER-3 antibodiescould reduce HRG-induced cell migration. See, FIG. 11 of US PublicationNo. 20080124345.

Example 18 Colony Formation Assay (Soft Agar Assay)

Soft agar assays were conducted in order to investigate the ability ofthe anti-HER-3 antibodies to inhibit anchorage independent cell growth.The soft agar colony formation assay is a standard in vitro assay totest for transformed cells, as only such transformed cells can grow insoft agar.

750 to 2000 cells (depending on the cell line) were preincubated withindicated antibodies at 10 μg/ml in IMDM medium (Gibco) for 30 min andresuspended in 0.4% Difco noble agar. The cell suspension was plated on0.75% agarose underlayer containing 20% FCS in quadruplicate in a96-well plate. Colonies were allowed to form for 14 days, and were thenstained with 50 μl MTT (0.5 mg/ml in PBS) overnight, and counted with aScanalyzer HTS camera system (Lemnatec, Wuerselen). Anti-HER-3antibodies were able to reduce anchorage independent cell growth ofMDA-MB361 and NCI-ADR breast cancer cells, MKN-28 gastric cancer cells,HT144 melanoma cells, Skov3 ovary carcinoma cells, PPC-1 prostate cancercells, BX-PC3 pancreas cancer cells, A431 epidermoid carcinoma cells,and lung carcinoma cells. See, FIGS. 12a-12i of US Publication No.20080124345.

EXAMPLE 19 Human Anti-HER-3 Antibodies Inhibit Human Breast CarcinomaGrowth in Nude Mice

The anti-tumor efficacy of therapeutic antibodies is often evaluated inhuman xenograft tumor studies. In these studies, human tumors grow asxenografts in immunocompromised mice and therapeutic efficacy ismeasured by the degree of tumor growth inhibition. In order todetermine, if the anti-HER-3 antibodies interfere with tumor growth ofhuman breast cancer cells in nude mice, 5×10⁶ T47D cells were implantedin female NMRI nude/nude mice. Tumors were subcutaneous, grown on theback of the animal. Treatments began when tumors reached a mean volumeof 20 mm³; eight days post implantation. Prior to first treatment, micewere randomized and statistical tests performed to assure uniformity instarting tumor volumes (mean, median and standard deviation) acrosstreatment groups. Treatment started with a loading dose of 50 mg/kgfollowed by 25 mg/kg injections once a week by intraperitonealinjection. A control arm received doxorubicin (pharmaceutical grade).All animals were supplemented with 0.5 mg/kg/week oestrogen injectedi.p. Details of the treatment groups are given in TABLE 8 below. Thesestudies demonstrated that administration of an anti-HER-3 antibodyresulted in reduction of tumor growth. See, FIG. 13 of US PublicationNo. 20080124345.

TABLE 8 Loading Weekly dose Gr. N 1^(st) Compound (mg/kg) (mg/kg) RouteSchedule 1. 10 PBS — i.p. once/week 2. 10 Doxorubicin  8 mg/kg i.v.once/week* 3. 10 U1-53 50 mg/kg 25 mg/kg i.p. once/week 20 ml/kg 10ml/kg *doxorubin treatment as described by Boven et al., CancerResearch, 1992.

Example 20 Human Anti-HER-3 Antibodies Inhibit Human Pancreatic TumorGrowth in SCID Mice

To test the therapeutic potential of anti-HER-3 antibodies in othersolid tumor types the anti-HER-3 antibodies, U1-53 and U1-59, weretested in mice with established tumors derived from the human pancreatictumor cell line BxPC3. As controls sets of mice treated with either thevehicle control, PBS, or the established therapeutic antibody, Erbitux,were included. 5×10⁶ BxPC3 cells were inoculated subcutaneously withoutMatrigel into CB17 SCiD mice. Mice bearing established tumors with amean volume of 140 mm² received 50 mg/kg of U1-53, U1-59, Erbitux or theequivalent volume of PBS via intraperitoneal injection. Thereafter themice received once weekly 25 mg/kg injections for the duration of thestudy.

U1-53 and U1-59 reduced the growth of the human pancreatic tumors in acytostatic fashion. See, FIG. 14 of US Publication No. 20080124345.Notably, in this experiment, U1-53 and U1-59 were more effective thanthe EGF-R-targeting antibody Erbitux at delaying tumor growth. Thesestudies demonstrated the therapeutic efficacy of anti-HER-3 antibodiesin comparison to a benchmark therapeutic agent.

Example 21 Combining the Human Anti-HER-3 Antibodies with Anti-EGF-RAntibodies Increases Anti-Tumor Activity

The monotherapy of hyperproliferative diseases with targeted antibodiesis often hampered by problems such as, on the one hand, the developmentof resistance to drugs, and on the other hand, a change in theantigenicity. For example, loss of antigenicity after prolongedtreatment may render tumor cells insensitive to therapeutic antibodies,since those tumor cells that do not express or have lost the targetedantigen have a selective growth advantage. These problems might beevaded by using the antibodies in combination with a therapeuticantibody that targets a different receptor on the tumor cells, oranother antineoplastic agent. Intervening in multiple signaling pathwaysor even related pathways but at multiple intervention steps might alsoprovide therapeutic benefit. These combined treatment modalities arelikely to be more efficacious, because they combine two anti-canceragents, each operating via a different mechanism of action.

In order to demonstrate the feasibility of the anti-HER-3 antibodies U1-53 and U1-59 as suitable combination agents, we comparedmonotherapeutic administrations of U1-53 or U1-59 with those in whicheither U1-53 or U1-59 was combined with the anti-EGR specific antibody,Erbitux. 5×10⁶ BxPC3 cells were inoculated subcutaneously with Matrigelinto CB17 SCID mice. After tumor volumes had reached 200 mm³, mice wererandomized into individual treatment groups. Weekly intraperitonealadministrations of U1-53, U1-59 and Erbitux as single agents orcombinations of either anti-HER-3 antibodies with Erbitux or as acocktail of two anti HER-3 antibodies were performed. All antibodieswere dosed at a single loading dose of 50 mg/kg/week, followed by weeklyinjections of 25 mg/kg for six weeks. Control arms received bi-weeklyadministrations of Gemcitabine (120 mg/kg), weekly pooled human IgG orweekly vehicle (PBS) injections. The regimens are detailed in TABLE 9below.

TABLE 9 Loading dose weekly dose Gr. N Compound (mg/kg) (mg/kg) RouteSchedule 1 12 PBS 20 ml/kg 10 ml/kg q7d i.p. 2 12 Pooled human IgG 50mg/kg 25 mg/kg q7d i.p. 3 12 U1-53 50 mg/kg 25 mg/kg q7d i.p. 4 12 U1-5950 mg/kg 25 mg/kg q7d i.p. 5 12 Erbitux 50 mg/kg 25 mg/kg q7d i.p. 6 12U1-53 + Erbitux 25 mg/kg each 12.5 mg/kg each q7d i.p. 7 12 U1-59 +Erbitux 25 mg/kg each 12.5 mg/kg each q7d i.p. 8 12 U1-53 + U1-59 25mg/kg each 12.5 mg/kg each q7d i.p. 9 12 Gemcitabine none 120 mg/kg 2xweekly i.p.

Antibodies U 1-53 and U 1-59, when administered as single agents,delayed the growth of the human pancreatic tumors to the same degree asGemcitabine, which is often used as a standard anti-pancreatic cancerchemotherapy. Co-administration of Erbitux with U1-53 or U1-59 resultedin a significantly greater reduction of tumor growth than observed witheither single agent administration of U1-53, U1-59 or Erbitux. Thus, abeneficial therapeutic response can be achieved by combining theanti-HER-3 antibodies with suitable antibodies that target separatetumor antigens. See, FIG. 15 of US Publication No. 20080124345.

In summary, the anti-HER-3 antibodies had potent therapeutic efficacyagainst human tumors in vivo. They can be effectively combined withother anti-neoplastic therapeutics for increased anti-tumor activity.

Example 22 Human Anti-HER-3 Antibodies Inhibit Human Melanoma TumorGrowth in nu/nu Mice

Members of the erbB family of receptors, including HER-3, are abnormallyexpressed in a large variety of epithelial cancers and they are known toplay important roles in the growth and survival of many these solidtumors. These tumors include melanomas, head and neck squamous cellcancers, non-small cell lung cancers and prostate, glioma, gastric,breast, colorectal, pancreatic, ovarian cancers. In order to verify,that the anti-HER-3 antibodies are not restricted in their anti-canceractivity to individual tumor types, e.g., pancreatic cancers (see,Example 21), but can be used as therapeutics against manyHER-3-dependent tumors, we tested U1-53 and U1-59 in additionalxenograft studies. Human melanoma cells (5×10⁵), HT144, were injectedsubcutaneously into CB17 SCID mice, followed by immediate subsequentintraperitoneal injection of 50 mg/kg of U1-53 and U1-59, the equivalentvolume of PBS or Dacarbacin (DITC) at 200 mg/kg. Thereafter, micereceived 25 mg/kg of U1-53 or U1-59 once weekly, whereas DITC was givenonce every two weeks at 200 mg/kg.

The median tumor volumes from each treatment group were calculated.Administration of the antibodies resulted in growth reduction of thehuman melanomas when compared to tumors that had been treated with thevehicle control. See, FIG. 16 of US Publication No. 20080124345. Theseresults demonstrate that the antibodies are not restricted in theirtherapeutic potential and target a wide variety of HER-3 expressingcancers.

Example 23 Human Anti-HER-3 Antibodies Inhibit Growth of Colon CarcinomaXenografts in Mice

HT-29 human colon carcinoma cells were suspended in medium with 2:1ratio of Matrigel to a final concentration of 10×10⁶ cells/ml. 0.2 ml ofcell suspension were injected s.c. into the right flank of 4-5-week-oldCD1 nu/nu mice. A total of 95 mice were used.

The mice were randomly assigned to control and treatment groups. Thetreatment started on the same day. Duration of treatment was 29 days.Upon completion of the study, three tumors per group were collected 3hours after administration of treatment. The tumours were fast-frozenand kept at −80° C.

The following treatment protocol was carried out:

-   -   Control group: non-specific human IgG 25 mg/kg, twice weekly,        intraperitoneal    -   Treatment group: antibody U1-53, 25 mg/kg, twice weekly,        intraperitoneal    -   Treatment group: antibody U1-7, 25 mg/kg, twice weekly,        intraperitoneal    -   Treatment group: antibody U1-59, 25 mg/kg, twice weekly,        intraperitoneal    -   Treatment group 5-FU: 5-fluorouracil, 50 mg/kg, 9 d×5,        intraperitoneal

The median tumor volumes from each group were calculated. Administrationof the antibodies resulted in growth reduction of the HT-29 coloncarcinoma tumors when compared to tumors that had been treated withnon-specific human IgG1. See, FIG. 17 of US Publication No. 20080124345.

Example 24 Human Anti-HER-3 Antibodies Inhibit Lung Cancer Growth inMice

Calu-3 human non-small cell lung cancer cells were suspended in mediumwith 1:1 ratio of Matrigel to a final concentration of 5×10⁶ cells/ml.0.05 ml of cell suspension were injected s.c. into the right flank of9-week-old female CB17 scid mice. A total of 60 mice were used.

The mice were randomly selected to control and treatment groups.Treatment started on the same day. The duration of treatment was 32days.

The following treatment protocol was carried out:

PBS Vehicle Group

-   -   hG control group: non-specific human IgG: 25 mg/kg, twice        weekly, intraperitoneal    -   Treatment group antibody U1-53, 25 mg/kg, twice weekly,        intraperitoneal    -   Treatment group antibody U1-7, 25 mg/kg, twice weekly,        intraperitoneal    -   Treatment group antibody U1-59, 25 mg/kg, twice weekly,        intraperitoneal

The median tumor volumes from each control and treatment group werecalculated. Administration of the antibodies resulted in growthreduction of the human non-small lung cancer xenografts when compared totumors that had been treated with the PBS vehicle control ornon-specific human IgG. See, FIG. 18 of US Publication No. 20080124345.

Example 25 Human Anti-HER-3 Antibodies Inhibit Human Pancreatic TumorGrowth in Balb/C-Mice

Human pancreatic BxPC3 tumor cells were suspended in medium with a 2:1ratio of Matrigel to a final concentration of 5×10⁶ cells per ml. 0.2 mlof cell suspension were injected s.c. into the right flank of5-7-week-old female BalbC nu/nu mice. A total of 100 mice were used.

The mice were randomly distributed into control and treatment groups.The treatment started on the same day. The treatment duration was 27days.

The following treatment protocol was carried out:

-   -   hIgG control group: non-specific human IgG2, 25 mg/kg, twice        weekly, intraperitoneal    -   Treatment group antibody U1-53, 25 mg/kg, twice weekly,        intraperitoneal    -   Treatment group antibody U1-7, 25 mg/kg, twice weekly,        intraperitoneal    -   Treatment group antibody U1-59, 25 mg/kg, weekly,        intraperitoneal    -   Gemzar treatment group, gemcitabine, 80 mg/kg, weekly,        intraperitoneal

The median tumor volumes from each control and treatment group werecalculated. Administration of the antibodies resulted in growthreduction of the human pancreatic tumors when compared to tumors thathad been treated with non-specific human IgG or with Gemzar. See, FIG.19 of US Publication No. 20080124345.

The inhibition of HER-3 in the human pancreatic tumors could also beshown in a pharmacodynamic experiment. The BxPC3 tumor xenografts weregrown as described above. 3 mice were treated with 500 μg of an IgG1control antibody and 3 mice were treated with 500 μg of the anti-HER-3antibody U1-59. The mice were treated on day 1 and day 4 and thensacrificed on day 5 to measure the antibody-dependent inhibition ofHER-3 phosphorylation (pHER-3).

The tumors were homogenized in a standard RIPA buffer with proteaseinhibitors. 50 μg clear lysate was separated on a 4-20% Tris-glycinegel, transferred onto a nitrocellulose membrane and blocked in 3% bovineserum albumin (BSA). Immunoblotting was performed using an anti-pHER-3antibody (antibody 21D3, Cell Signaling technology). An anti-actinantibody (AB a-2066, Sigma) was used as a control.

Expression was detected by enhanced chemiluminescence (AmershamBiosciences, Piscataway, N.J.). The images were captured with theVersadoc 5000 Imaging System (BioRad, Hercules, Calif.). Afteradministration of the human anti-HER-3-antibody U1-59, phosphorylationof HER-3 was no longer detectable. See, FIG. 20 of US Publication No.20080124345. Thus, the antibodies were capable of significantly reducingHER-3 activation in human pancreatic tumor cells.

Example 26 U1-59 Inhibits Tumor Growth in Combination with a SecondAgent in Xenograft Studies

Calu-3 NSCLC tumor xenograft models were used to evaluate theeffectiveness of an anti-HER-3 antibody (U1-59), either alone or incombination with panitumamab or erlotinib. To determine in vivoefficacy, mice bearing ˜200 mm³ Calu-3 NSCLC xenografts were treatedtwice a week with anti-HER family inhibitors or control. Otherexperiments were done with A549 cells. In the combination studies withpanitumumab, IgG1 was used as a negative control for U1-59, and IgG2 wasused as a negative control for panitumumab. As shown in FIG. 1, while100 μg of U1-59 or 100 μg of panitumumab alone greatly reduced tumorgrowth as compared to control, the combination of 100 μg of each of thetwo agents completely inhibited tumor growth (p<0.0001 for thecombination vs. either agent alone). In the combination studies witherlotinib, IgG1 was used as a negative control for U1-59, and erlotinibvehicle was used as a negative control for erlotinib. As shown in FIG.2, the combination of 100 μg U1-59 and 25 μg erlotinib had a greaterinhibitory effect than either agent alone. The combination of UI-59 witherlotinib was significantly more effective than U1-59 alone (p=0.0376).

Example 27 U1-59 in Combination with HER Inhibitors InhibitsAnchorage-Independent Growth of Breast and Ovarian Cancer Cells

Experiments were conducted to evaluate the effect of U1-59 incombination with the HER inhibitors pertuzumab, trastuzumab, orcetuximab on anchorage-independent growth of SkBr-3 (basal or HRGstimulated) and MDA-MB-435 (basal) cancer cells. IgG was used as anegative control for all studies. Tumor cell colonies formed in theabsence or presence of HRG for 6 to 10 days and were stained with MTTfor 4 to 6 hours and quantified. U1-59 as a single agent did not inhibitcolony growth of MDA-MB 435 cells, but inhibited colony growth by 50% inthe SkBr-3 cells (p<0.001), and up to 95% when combined with other HERinhibitors (p<0.05). For example, the combination of 5 μg/ml pertuzumabor trastuzumab with 5 μg/ml U1-59 reduced anchorage-independent growthin basal SkBr-3 breast cancer cells significantly more than either agentalone (FIG. 3), pertuzumab, trastuzumab, or cetuximab in combinationwith U1-59 were significantly (p<0.006) more effective than U1-59 alonein HRG stimulated SkBr-3 cells (FIG. 4). Similarly, combinations ofU1-59 with either pertuzumab, trastuzumab or cetuximab inhibited colonyformation of basal ovarian cancer cells (MDA-MB-435) significantlybetter (p<0.002) than U1-59 alone (FIG. 5).

Example 28 U1-59 in Combination with HER-2 Inhibitors orChemotherapeutic Agents Reduces Cancer Cell Proliferation

Studies were conducted to evaluate the effect of U1-59 in combinationwith HER-2 inhibitors or chemotherapeutic agents on cancer cellproliferation. In particular, the following experiments were conductedin MDA-MB-175 VII breast cancer cells:

-   -   U1-59 and Trastuzumab        -   Control=DMSO+75 μg/ml IgG1+PBS        -   10 μg/ml U1-59        -   75 μg/ml Trastuzumab        -   10 μg/ml U1-59+75 μg/ml Trastuzumab    -   U1-59 and Lapatinib        -   Control=DMSO+150 μg/ml IgG1        -   73.5 μg/ml U1-59        -   0.1 μM Lapatinib        -   73.5 μg/ml U1-59+0.1 μM Lapatinib    -   U1-59 and Gemcitibine        -   Control=DMSO+75 μg/ml IgG1+PBS        -   10 μg/ml U1-59        -   1 μg/ml Gemcitibine        -   10 μg/ml U1-59+1 μg/ml Gemcitibine    -   U1-59 and Cisplatin        -   Control=DMSO+75 μg/ml IgG1+PBS        -   10 μg/ml U1-59        -   1 μg/ml Cisplatin        -   10 μg/ml U1-59+1 μg/ml Cisplatin

MDA-MB-175VII breast cancer cells were incubated with U1-59 and/or theother agents for 1 hour prior to HRG stimulation. After four days, thegrowth of treated cells was measured with ALOMAR BLUE™. In these assays,U1-59 reduced HRG-stimulated MDA-MB-175VII proliferation up to 40%(p<0.05) as a single agent, and up to 80% (p<0.05) when combined withtrastuzumab or lapatinib (FIGS. 6A and 6B). Of note, additive activityalso was observed in MDA-MB-175VII cells when U1-59 was combined withstandard of care chemotherapeutics (gemcitabine and cisplatin; p<0.05vs. either single agent alone) (FIGS. 6C and 6D). In each of theseexperiments, the combination of U1-59 with the HER-2 inhibitor was moreeffective at reducing proliferation of MDA-MB175VII cells than eitheragent alone.

Similar experiments were conducted with U1-59 and pertuzumab,trastuzumab, or lapatinib in HRG stimulated ZR-75-30 breast cancer cellsand HRG stimulated BT474 breast cancer cells (FIGS. 7 and 8,respectively). In each case, the combination of U1-59 and lapatinib hadthe greatest inhibitory effect on cell proliferation. Compared to singleagent treatment alone, the combination of U1-59 with pertuzumab ortrastuzumab or lapatinib was significantly (p<0.004) more effective thanU1-59 alone. Combining U1-59 with one or more of pertuzumab,trastuzumab, and cetixumab in HRG stimulated DLD-1 colon cancer cellsand HRG stimulated HCC-1569 breast cancer cells had similar effects, asshown in FIGS. 9 and 10. In addition, combinations of U1-59 withtrastuzumab or lapatinib in HRG stimulated SkBr-3 breast cancer cellsalso were more effective than U1-59 alone (p<0.004) (FIG. 11).

In additional experiments, Head and Neck cancer cells (FaDu) werecultured in growth medium (MEM+10% FBS+1× PSG) and treated with IgGcontrols, U1-59, panitumumab or a combination of U1-59 with panitumab.After incubation for 5 days at 37° C., proliferation was measured withALOMAR BLUE™. As a single agent, U1-59 reduced proliferation of FaDucells by 15% to 20%, whereas the combination of U1-59 with panitumumabresulted in more than 80% reduction. The combination of U1-59 withpanitumumab resulted in a significant (p=0.001 vs. best single agentactivity) improvement over the use of either agent alone (FIG. 12).

Example 29 U1-59 in Combination with Other HER Inhibitors InhibitsSignal Transduction

The effect of U1-59 either alone or in combination with cetuximab,pertuzumab, trastuzumab, or lapatinib on signal transduction wasmeasured in unstimulated MDA-MB-175VII breast cancer cells, HRGstimulated SkBr-3 breast cancer cells, HRG stimulated Ls174T coloncancer cells, and HRG stimulated HCC-1569 breast cancer cells. Cellswere treated with agents as indicated in FIGS. 13-16, andphosphorylation of HER-3, Akt, and ERK was evaluated by Western blottingwith phospho-specific antibodies. The combination of U1-59 with eitherpertuzumab, trastuzumab, or lapatinib further reduced phosphorylation ofHER-3, Akt and ERK in all cell types tested as compared to single agenttreatments. The combination of U1-59 with cetuximab appeared tosynergize less efficiently in these assays.

Similar studies were conducted in A549 alveolar epithelial cells (FIG.17) and Calu3 NSCLC cells (FIG. 18) treated with U1-59 alone or U1-59 incombination with panitumumab or lapatinib, using Western blotting toevaluate phosphorylation of Akt, EGF-R, HER-2, HER-3, HER-4, and ERK.The combination of U1-59 with panitumumab had the greatest apparenteffect on HER-3 phosphorylation in A549 cells, while the combination wasmore effective with regard to Akt and EGF-R phosphorylation in Calu3cells.

Additional experiments were conducted to evaluate the in vitro efficacyand anchorage-independent growth of A549 cells treated with 10 μg/mLU1-59, other HER family Abs, or control mAb in serum containing medium.Tumor cell colonies formed in the absence of exogenous ligand for 10days and were stained with MTT and quantified using a Scanalyzer HTScamera imaging system. U1-59 inhibited colony growth by 50% (p<0.001) inthe A549 cell line and resulted in tumor stasis in the A549 NSCLCxenograft model vs. IgG control or other HER mAbs (p<0.05).

These results demonstrate that U1-59 inhibits proximal and distal HER-3oncogenic signaling in breast cell lines in vitro, and that breastcancer cells are sensitive to U1-59 treatment as a single agent and incombination with anti-HER agents.

Example 30 U1-59 Sensitizes Lapatinib for in Vivo Activity

To evaluate the combined effects of U1-59 and lapatinib in vivo, micewere implanted with human breast cancer cells (HCC-1569) and treatedwith U1-59 and lapatinib either alone or in combination. Tumors wereallowed to reach sizes greater than or equal to 100 mm³, and mice weresubsequently treated with control, lapatinib, U1-59, or a combination ofU1-59 and lapantinib. As shown in FIG. 19, U1-59 alone did not inhibitHCC-1569 tumor growth, and lapatinib alone caused some, but notsignificant tumor growth inhibition compared to the control (p=0.16).The combination of lapatinib with U1-59, however, resulted insignificant inhibition of tumor growth (p<0.02 vs. control or p<0.05 vs.lapatinib).

These results indicate that the combination of U1-59 and lapatinibresulted in synergistic inhibition of HCC-1569 tumor growth in vivo.This result is particularly interesting and encouraging as it shows thateven tumor types that may not respond to U1-59 or lapatinib alone, canbe very effectively treated with the combination of both.

Example 31 Use of Anti-HER-3 Antibodies as Diagnostic Agents

Anti-HER-3 mAb can be used in the diagnostic of malignant diseases.HER-3 is expressed on tumor cells in a very distinct way compared tonormal tissue and, therefore, an expression analysis of HER-3 wouldassist in the primary diagnosis of solid tumors, staging and grading ofsolid tumors, assessment of prognostic criteria for proliferativediseases and neoplasias and risk management in patients with HER-3positive tumors.

A. Detection of HER-3 Antigen in a Sample

An Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of HER-3antigen in a sample is developed. In the assay, wells of a microtiterplate, such as a 96-well microtiter plate or a 384-well microtiterplate, arc adsorbed for several hr with a first fully human monoclonalantibody directed against the HER-3 antigen. The immobilized antibodyserves as a capture antibody for any of the HER-3 antigen that may bepresent in a test sample. The wells are rinsed and treated with ablocking agent such as milk protein or albumin to prevent nonspecificadsorption of the analyte.

Subsequently the wells are treated with a test sample suspected ofcontaining the HER-3 antigen, or with a solution containing a standardamount of the HER-3 antigen. Such a sample is, for example, a serumsample from a subject suspected of having levels of circulating HER-3antigen considered to be diagnostic of a pathology. After rinsing awaythe test sample or standard, the wells arc treated with a second fullyhuman monoclonal anti-HER-3 antibody that is labelled by conjugationwith biotin. The labeled anti-HER-3 antibody serves as a detectingantibody. After rinsing away excess secondary antibody, the wells aretreated with avidin-conjugated horseradish peroxidase (HRP) and asuitable chromogenic substrate. The concentration of the HER-3 antigenin the test samples is determined by comparison with a standard curvedeveloped from the standard samples.

B. Detection of HER-3-Antigen in Immunohistochemistry (IHC)

In order to determine HER-3-antigen in tissue sections by IHC,Paraffin-embedded tissues are first deparaffinized in xylene for 2×5 minand then hydrated with 100% Ethanol 2×3 min, 95% Ethanol 1 min andrinsed in distilled water. Antigenic epitopes masked byformalin-fixation and paraffin-embedding are exposed by epitopeunmasking, enzymatic digestion or saponin. For epitope unmaskingparaffin sections are heated in a steamer, water bath or microwave ovenfor 20-40 min in a epitope retrieval solution as for example 2N HClsolution (pH 1.0). In the case of an enzyme digestion, tissue sectionsare incubated at 37° C. for 10-30 minutes in different enzyme solutionssuch as proteinase K, trypsin, pronase, pepsin etc.

After rinsing away the epitope retrieval solution or excess enzyme,tissue sections are treated with a blocking buffer to prevent unspecificinteractions. The primary antibody is incubated at appropriate dilutionsin dilution buffer for 1 hour at room temperature or overnight. Excessprimary antibody is rinsed away and sections are incubated in peroxidaseblocking solution for 10 min at room temperature. After another washingstep, tissue sections are incubated with a secondary antibody labelledwith a group that might serve as an anchor for an enzyme. Examplestherefore are biotin labelled secondary antibodies that are recognizedby streptavidin coupled horseradish peroxidase. Detection of theantibody/enzyme complex is achieved by incubating with a suitablechromogenic substrate.

C. Determination of HER-3 Antigen Concentration in Serum of Patients

A sandwich ELISA is developed to quantify HER-3 levels in human serum.The two fully human monoclonal anti-HER-3 antibodies used in thesandwich ELISA, recognized different domains on the HER-3 molecule anddo not compete for binding, for example (see, Example 8). The ELISA isperformed as follows: 50 μl of capture anti-HER-3 antibody in coatingbuffer (0.1 M NaHCO₃, pH 9.6) at a concentration of 2 μg/ml were coatedon ELISA plates (Fisher). After incubation at 4° C. overnight, theplates are treated with 200 μl of blocking buffer (0.5% BSA, 0.1% Tween20, 0.01% Thimerosal in PBS) for 1 hr at 25° C. The plates were washed(3×) using 0.05% Tween 20 in PBS (washing buffer, WB). Normal or patientsera (Clinomics, Bioreclaimation) are diluted in blocking buffercontaining 50% human serum. The plates are incubated with serum samplesovernight at 4° C., washed with WB, and then incubated with 100 μl/wellof biotinylated detection anti-HER-3 antibody for 1 hr at 25° C. Afterwashing, the plates are incubated with HRP-Streptavidin for 15 min,washed as before, and then treated with 100 μl/well ofo-phenylenediamine in H₂O₂ (Sigma developing solution) for colorgeneration. The reaction is stopped with 50 μl/well of H₂SO₄ (2 M) andanalyzed using an ELISA plate reader at 492 nm. The concentration ofHER-3 antigen in serum samples is calculated by comparison to dilutionsof purified HER-3 antigen using a four parameter curve fitting program.

Staging of cancer in a patient: Based on the results set forth anddiscussed under items A, B and C, it is possible to stage a cancer in asubject based on expression levels of the HER-3 antigen. For a giventype of cancer, samples of blood are taken from subjects diagnosed asbeing at various stages in the progression of the disease, and/or atvarious points in the therapeutic treatment of the cancer. Theconcentration of the HER-3 antigen present in the blood samples isdetermined using a method that specifically determines the amount of theantigen that is present. Such a method includes an ELISA method, such asthe method described under items A and B. Using a population of samplesthat provides statistically significant results for each stage ofprogression or therapy, a range of concentrations of the HER-3 antigenthat may be considered characteristic of each stage is designated.

In order to stage the progression of the cancer in a subject understudy, or to characterize the response of the subject to a course oftherapy, a sample of blood is taken from the subject and theconcentration of the HER-3 antigen present in the sample is determined.The concentration so obtained is used to identify in which range ofconcentrations the value falls. The range so identified correlates witha stage of progression or a stage of therapy identified in the variouspopulations of diagnosed subjects, thereby providing a stage in thesubject under study.

Anti-HER-3 antibodies as described herein are used for treatment ofcertain hyperproliferative or HER-3 associated disorders based on anumber of factors, such as HER-3 expression, for example. Tumor typessuch as breast cancer, gastrointestinal cancer, pancreatic cancer,prostate cancer, ovarian cancer, stomach cancer, endometrial cancer,salivary gland cancer, lung cancer, kidney cancer, colon cancer,colorectal cancer, thyroid cancer, bladder cancer, glioma, melanoma, andother HER-3 expressing or overexpressing cancers are examples ofindications that are treated with a combination therapy as describedherein, although indications are not limited to those in the precedinglist. In addition, the following groups of patients may benefit fromtreatment as described herein:

-   -   Patients not eligible for treatment with anti-HER-2 mAb    -   Patients with resistance to anti-HER-1 mAb or small molecule        anti-EGF-R inhibitor    -   Patients with NSCLC resistant to erlotinib or gefitinib

Anti-HER-3 antibodies are used in combination with one or moreadditional agents in a so called “combination therapy.” Such combinationtherapy includes, but is not limited to, the agents disclosed herein.Combination therapy with anti-HER-3 antibodies and other agents canextend patient survival, increase time to tumor progression, or enhancequality of patient life. Protocol and administration design will addresstherapeutic efficacy as well as the ability to reduce the usual doses ofstandard therapies, such as chemotherapy or radiation therapy, forexample.

Treatment of humans with anti-HER-3 antibodies: To determine the in vivoeffects of anti-HER-3 antibody treatment in human patients with tumors,such human patients are injected over a certain amount of time with aneffective amount of anti-HER-3 antibody. At periodic times during thetreatment, the human patients are monitored to determine whether theirtumors progress, in particular, whether the tumors grow and metastasize.

A tumor patient treated with the anti-HER-3 antibodies has a lower levelof tumor growth and/or metastasis compared to the level of tumor growthand metastasis in tumor patients treated with the current standard ofcare therapeutics.

Treatment with anti-HER-3 antibody conjugates: To determine the in vivoeffects of anti-HER-3 antibody conjugates, human patients or animalsexhibiting tumors are injected over a certain amount of time with aneffective amount of anti-HER-3 antibody conjugate. For example, theanti-HER-3 antibody conjugate administered is DM1-anti-HER-3 antibodyconjugate, an auristatin-anti-HER-3 antibody conjugate orradioisotope-anti-HER-3 antibody conjugate. At periodic times during thetreatment, the human patients or animals are monitored to determinewhether their tumors progress, in particular, whether the tumors growand metastasize.

A human patient or animal exhibiting tumors and undergoing treatmentwith, for example, DM1-anti-HER-3 antibody or radioisotope-anti-HER-3antibody conjugates has a lower level of tumor growth and metastasiswhen compared to a control patient or animal exhibiting tumors andundergoing treatment with an alternate therapy. Control DM1-antibodiesthat may be used in animals include conjugates comprising DM1 linked toantibodies of the same isotype of the anti-HER-3 antibodies, but morespecifically, not having the ability to bind to HER-3 tumor antigen.Control radioisotope-antibodies that may be used in animal tests includeconjugates comprising radioisotope linked to antibodies of the sameisotype of the anti-HER-3 antibodies, but more specifically, not havingthe ability to bind to HER-3 tumor antigen. Note: the control conjugateswould not be administered to humans.

Example 33 Identifying First in Human Doses and Schedule of anAnti-HER-3 mAb Based on Preclinical Pharmacokinetic, Pharmacodynamic,and Efficacy Data

Studies were conducted to use preclinical modeling to predict aminimally effective dose regimen for objective response usingpreclinical pharmacokinetics (PK), BxPC3 xenograft mice anti-tumorefficacy, and pharmacodynamic (PD) data.

Mice bearing ˜200 mm³ established BxPC3 pancreatic xenografts weretreated twice per week with U1-59 at 25, 100, 200, 500 μg/mouse.Inhibition of pHER in the BxPC3 xenograft tumors was analyzed by westernblotting. A PK/PD/Efficacy model (based on Simeoni et al. (2004) CancerRes. 64:1094-1101) was used to prospectively select dose and schedulefor further testing. To confirm the PK/PD/Efficacy model, BxPC3pancreatic tumor-bearing mice were treated with 400 μg/mouse biweeklyand 200 μg/mouse biweekly, weekly and twice a week. Interspecies scalingbased on body weight (BW) was used to predict U1-59 PK parameters inhuman on the basis of the serum concentrations obtained in mice, rat andmonkeys. The relationship between drug concentration, inhibition ofpHER-3 in animals, and interspecies PK scaling was used to select theminimally effective dose for the first in human study.

U1-59 treatment of BxPC3 xenografts resulted in a statisticallysignificant inhibition of tumor growth and pHER-3 levels in a dose andschedule dependent manner (p<0.05). Treatment with U1-59 at 400 μg/mousebiweekly and 200 μg/mouse biweekly, weekly and twice a week resulted ina 50%, 33%, 74% and 70% inhibition of tumor growth (p<0.05), a 30%, 58%,23% and 20% inhibition of pHER-3 (quantitative Western blot) versus theIgG control treated group, respectively. Serum concentrations of U1-59at necropsy for the respective dose groups were (mean (SD)) of 2.07(0.97), 0.45 (0.21), 3.08 (0.82) and 34.9 (9.1) μg/mL, respectively. Theestimated trough concentration needed to achieve 90% maximal pHER-3inhibition (IC₉₀) was estimated to be ˜3 μg/ml. The PK/PD/efficacy modeldeveloped predicted the mean tumor volume (R²=0.925). The clearance (CL)and initial volume of distribution (Vd) in man were estimated to be 11mL/day/kg and 28 mL/kg. Comparison of simulated human PK profilessuggested that biweekly doses of >3 mg/kg, which should exhibit linearPK, may result in >90% pHER-3 inhibition during two week dosinginterval.

The anti-tumor efficacy in the BxPC3 pancreatic xenograft model wascorrelated with an increased serum concentration of U1-59 and a decreasein pHER-3 levels, allowing for development of a PK/PD/Efficacyrelationship. This relationship was used to determine a dose andschedule for U1-59 to investigate in a first in human (FIH) study.

Example 34 Reactivation Studies

A549 cells were plated in Ham's F-12 medium (Gibco), all mediasupplemented with 10% FBS (Hyclone, Logan, Utah) and 1× L-glutamine(Gibco). Cells were serum-starved overnight. The media were changed intofresh serum-free media and cells were treated with 50 μg/ml U1-59 or 5μM gefitinib alone, or combination of U1-59 and gefitinib, for 1 or 24hours at 37° C. Cells were washed with cold PBS after their respectivetreatment time points and lysed using RIPA buffer (20 mM Tris-HCl pH7.5, 1% Igepal, 1% sodium deoxycholate, 150 mM NaCl, 0.1% SDS, 1% TritonX-100) containing 200 μM phenylmethanesulfonylfluoride (PMSF) (FlukaBiochemica), 200 μM Halt protease inhibitor cocktail kit (PierceBiotechnology), and 200 μM sodium orthovanadate (Sigma-Aldrich, St.Louis, Mo.). The lysates were passed through QIA shredder columns(Qiagen) and the flow-through quantitated using a spectrophotometer(Beckman Coulter, Fullerton, Calif.). Proteins, 50 μg per well, wereanalyzed in duplicate for pHER3 using ELISA Duoset (R&D systems)according to manufacturer's protocol. The results arc shown in FIG. 20.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

TABLE 10 CDR Sequences AB- PAT SEQ SEQ SEQ Chain ID: ID: CDR1 ID: CDR2ID: CDR3 heavy U1-1 235 GGSINSGDY 258 YIYYSGSTY 283 ADYDFWSG YWS YNPSLKSYFDY light U1-1 318 RSQGIRNDA 343 AASSLQS 360 LQHNSYPWT LG heavy U1-2236 GGSISSGDY 259 YIYYSGSTY 283 ADYDFWSG YWS YNPSLRS YFDY light U1-2 318RASQGIRND 343 AASSLQS 361 LQHNGYPW LG T heavy U1-3 237 GGSISSGGY 258YIYYSGSTY 284 DGYDSSGY YWS YNPSLKS YHGYFDY light U1-3 319 KSSQSVLYS 344WASTRES 362 QQYYSTPLT SNNKNYLA heavy U1-4 236 GGSISSGDY 258 YIYYSGSTY283 ADYDFWSG YWS YNPSLKS YFDY light U1-4 318 RASQGIRND 343 AASSLQS 363LQHNNYPW LG T heavy U1-5 236 GGSISSGDY 258 YIYYSGSTY 283 ADYDFWSG YWSYNPSLKS YFDY light U1-5 318 RASQGIRND 343 AASSLQS 364 LQHNTYPW LG Theavy U1-6 236 GGSISSGDY 258 YIYYSGSTY 285 ADYDFWNG YWS YNPSLKS YFDYlight U1-6 318 RASQGIRND 343 AASSLQS 364 LQHNTYPW LG T heavy U1-7 236GGSISSGDY 258 YIYYSGSTY 283 ADYDFWSG YWS YNPSLKS YFDY light U1-7 320RASQDIRND 343 AASSLQS 360 LQHNSYPWT LG heavy U1-8 238 GYTLTELSM 260GFDPEDGETI 286 GWNYVFDY Y YAQKFQG light U1-8 321 RSSQSLLHS 345 LDSHRAS365 MQALQTPLT NGYNYLD heavy U1-9 236 GGSISSGDY 258 YIYYSGSTY 285ADYDFWNG YWS YNPSLKS YFDY light U1-9 320 RASQDIRND 343 AASSLQS 360LQHNSYPWT LG heavy U1-10 236 GGSISSGDY 258 YIYYSGSTY 283 ADYDFWSG YWSYNPSLKS YFDY light U1-10 318 RASQGIRND 343 AASSLQS 363 LQHNNYPW LG Theavy U1-11 236 GGSISSGDY 258 YIYYSGSTY 283 ADYDFWSG YWS YNPSLKS YFDYlight U1-11 318 RASQGIRND 343 AASSLQS 364 LQHNTYPW LG T heavy U1-12 236GGSISSGDY 258 YIYYSGSTY 283 ADYDFWSG YWS YNPSLKS YFDY light U1-12 318RASQGIRND 343 AASSLQS 363 LQHNNYPW LG T heavy U1-13 237 GGSISSGGY 258YIYYSGSTY 287 EDDGMDV YWS YNPSLKS light U1-13 322 RSSQSLLHS 346 LGSNRAS366 MQALQTPIT NGYNYLE heavy U1-14 236 GGSISSGDY 258 YIYYSGSTY 283ADYDFWSG YWS YNPSLKS YFDY light U1-14 318 RASQGIRND 343 AASSLQS 364LQHNTYPW LG T heavy U1-15 239 GGSVSSGGY 261 YIYYSGSTN 288 DGDVDTAM YWSYNPSLKS VDAFDI light U1-15 323 RASQSLSGN 347 GASSRAT 367 QQYDRSPLT YLAheavy U1-16 236 GGSISSGDY 258 YIYYSGSTY 283 ADYDFWSG YWS YNPSLKS YFDYlight U1-16 318 RASQGIRAD 343 AASSLQS 360 LQHNSYPWT LG heavy U1-17 236GGSISSGDY 262 YIYYSGSTY 283 ADYDFWSG YWS YNSSLKS YFDY light U1-17 318RASQGIRND 343 AASSLQS 360 LQHNSYPWT LG heavy U1-18 236 GGSISSGDY 258YIYYSGSTY 283 ADYDFWSG YWS YNPSLKS YFDY light U1-18 318 RSQGIRNDA 343AASSLQS 360 LQHNSYPWT LG heavy U1-19 236 GGSISSGDY 258 YIYYSGSTY 289GDYDFWSG YWS YNPSLKS EFDY light U1-19 sequence not available heavy U1-20237 GGSISSGGY 263 YIYDSGSTYY 290 DQGQDGYS YWS NPSLKS YGYGYYYG MDV lightU1-20 324 QASQDISNY 348 VASNLET 368 QQCDNLPLT LN heavy U1-21 236GGSISSGDY 258 YIYYSGSTYY 283 ADYDFWSG YWS NPSLKS YFDY light U1-21 320RASQDIRND 349 AASRLQS 360 LQHNSYPWT LG heavy U1-22 236 GGSISSGDY 258YIYYSGSTYY 283 ADYDFWSG YWS NPSLKS YFDY light U1-22 318 RASQGIRND 350AASSLQN 360 LQHNSYPWT LG heavy U1-23 236 GGSISSGDY 258 YIYYSGSTYY 283ADYDFWSG YWS NPSLKS YFDY light U1-23 318 RASQGIRND 343 AASSLQS 360LQHNSYPWT LG heavy U1-24 236 GGSISSGDY 258 YIYYSGSTYY 285 ADYDFWNG YWSNPSLKS YFDY light U1-24 318 RASQGIRND 343 AASSLQS 363 LQHNNYPW LG Theavy U1-25 236 GGSISSGDY 258 YIYYSGSTYY 283 ADYDFWSG YWS NPSLKS YFDYlight U1-25 318 RASQGIRND 350 AASSLQN 360 LQHNSYPWT LG heavy U1-26 236GGSISSGDY 258 YIYYSGSTYY 291 ADYDFWSG YWS NPSLKS YFDF light U1-26 318RASQGIRND 343 AASSLQS 361 LQHNGYPW LG T heavy U1-27 236 GGSISSGDY 258YIYYSGSTYY 291 ADYDFWSG YWS NPSLKS YFDF light U1-27 318 RASQGIRND 343AASSLQS 361 LQHNGYPW LG T heavy U1-28 236 GGSISSGDY 258 YIYYSGSTYY 292ADYDFWSG YWS NPSLKS YFDS light U1-28 318 RASQGIRND 343 AASSLQS 361LQHNGYPW LG T heavy U1-29 240 GFTFNSYDM 264 VIWYDGSNK 293 DRLCTNGVC HYYADSVKG YEDYGMDV light U1-29 324 QASQDISNY 351 DASNLET 369 QHYDTLPLT LNheavy U1-30 236 GGSISSGDY 265 YIYYSGTTYY 283 ADYDFWSG YWS NPSLKS YFDYlight U1-30 325 RAGQGIRND 343 AASSLQS 360 LQHNSYPWT LG heavy U1-31 241GYTFTNYGI 266 WISAYDGYR 294 DVQDYGDY S NYAQKLQG DYFDY light U1-31 326RASQSISSYL 343 AASSLQS 370 QQSYSTPIT N heavy U1-32 236 GGSISSGDY 265YIYYSGTTYY 283 ADYDFWSG YWS NPSLKS YFDY light U1-32 325 RAGQGIRND 343AASSLQS 360 LQHNSYPWT LG heavy U1-33 236 GGSISSGDY 258 YIYYSGSTYY 295ADYDFWSG YWS NPSLKS HFDC light U1-33 327 RASQGIRDD 352 AESSLQS 371LQHHSYPWT LG heavy U1-34 241 GYTFTNYGI 266 WISAYDGYR 294 DVQDYGDY SNYAQKLQG DYFDY light U1-34 326 RASQSISSYL 343 AASSLQS 370 QQSYSTPIT Nheavy U1-35 242 GFTFSDYYM 267 YISSSGNNIY 296 ERYSGYDDP S HADSVKG DGFDIlight U1-35 328 QASQDISNY 351 DASNLET 372 QQYDNPPCS LS heavy U1-36 243GGSISSGYY 268 YIYYSGTTYY 297 ADYDFWSG YWS NPSFKS HFDY light U1-36 318RASQGIRND 343 AASSLQS 360 LQHNSYPWT LG heavy U1-37 244 GYTFTSYGIS 269WISAYDGHT 298 DPHDYSNYE NYAQKLQG AFDF light U1-37 326 RASQSISSYL 343AASSLQS 370 QQSYSTPIT N heavy U1-38 245 GFSLSTSGV 270 LIYWNDDKR 299RDEVRGFDY GVG YSPSLKS light U1-38 329 RSSQSLVYS 353 KVSNWDS 373 MQGAHWPIDGYTYLH T heavy U1-39 246 GFTVSSNYM 271 VIYSGGSTYY 300 GQWLDV S ADSVKGlight U1-39 321 RSSQSLLHS 354 LGFHRAS 374 RQALQTPLT NGYNYLD heavy U1-40237 GGSISSGGY 272 YIYSSGSTYY 301 DRELELYYY YWS NPSLKS YYGMDV light U1-40330 RSSQSLLYS 346 LGSNRAS 365 MQALQTPLT NGYNYLD heavy U1-41 237GGSISSGGY 258 YIYYSGSTYY 302 DRELEGYSN YWS NPSLKS YYGVDV light U1-41 331RASQAISNY 343 AASSLQS 375 QQNNSLPIT LN  heavy U1-42 247 GYSFTSYWI 273IIYPGDSDTR 303 HENYGDYN G YSPSFQG Y light U1-42 332 RASQSIRSYL 343AASSLQS 376 QQSNGSPLT N  heavy U1-43 237 GGSISSGGY 259 YIYYSGSTYY 304DREREWDD YWS NPSLKS YGDPQGMD V light U1-43 333 RASQSISSYL 343 AASSLQS377 QQSYSNPLT H  heavy U1-44 247 GYSFTSYWI 274 IIWPGDSDTI 303 HENYGDYN GYSPSFQG Y light U1-44 332 RASQSIRSYL 343 AASSLQS 378 QQSISSPLT N  heavyU1-45 248 GYTFTSYDI 275 WMNPNSGDT 305 FGDLPYDYS N GYAQVFQG YYEWFDP lightU1-45 326 RASQSISSYL 343 AASSLQS 379 QQSYSTPLT N  heavy U1-46 249GDSVSSNSA 276 RTYYRSKWY 306 DLYDFWSG AWN NDYAVSVKS YPYYYGMD V lightU1-46 sequence not available heavy U1-47 249 GDSVSSNSA 276 RTYYRSKWY 307DYYGSGSFY AWN NDYAVSVKS YYYGMDV light U1-47 326 RASQSISSYL 355 AASNLQS380 QQSYSTPRT N heavy U1-48 250 GGSISSYYW 277 HIYTSGSTNY 308 EAIFGVGPY SNPSLKS YYYGMDV light U1-48 sequence not available heavy U1-49 251GYTFTGYY 278 WINPNIGGTN 309 GGRYSSSWS MH CAQKFQG YYYYGMDV light U1-49334 KSSQSLLLS 356 EVSNRFS 381 MQSMQLPIT DGGTYLY heavy U1-50 239GGSVSSGGY 261 YIYYSGSTNY 310 GGDSNYED YWS NPSLKS YYYYYGMD V light U1-50335 RASQSISIYL 343 AASSLQS 382 QQSYTSPIT H  heavy U1-51 250 GGSISSYYW261 YIYYSGSTNY 311 DSSYYDSSG S NPSLKS YYLYYYAM DV light U1-51 319KSSQSVLYS 344 WASTRES 383 QQYYTTPLT SNNKNYLA heavy U1-52 237 GGSISSGGY279 NIYYSGSTYY 312 GGTGTNYY YWS NPSLKS YYYGMDV light U1-52 336 RASQSVSSS357 GASSWAT 384 QQYGSSPLT YLA heavy U1-53 252 GFTFSIYSM 280 YISSSSSTIYY313 DRGDFDAFD N ADSVKG I light U1-53 337 QASQDITNY 351 DASNLET 385QQCENFPIT LN heavy U1-55.1 253 GGSVSSGGY 281 YINYSGSTNY 301 DRELELYYYYWN NPSLKS YYGMDV light U1-55.1 same as U1-55 heavy U1-55will be same as U1-55.1 light U1-55 338 RSSQSLLYS 346 LGSNRAS 366MQALQTPIT NGYKYLD heavy U1- 57.1 same as U1-57 light U1-57.1 338RSSQSLLYS 346 LGSNRAS 366 MQALQTPIT NGYKYLD heavy U1-57 254 GGSVSSGGY281 YINYSGSTN 301 DRELELYYY YWN YNPSLKS YYGMDV light U1-57will be same as U1-57.1 heavy U1-58 255 GFTFSSYGM 264 VIWYDGSNK 314AARLDYYY H YYADSVKG GMDV light U1-58 339 RASQSINSY 358 GASGLQS 386QQSYSSPLT LN  heavy U1-59 256 GGSFSGYY 282 EINHSGSTNY 315 DKWTWYFD WSNPSLKS L light U1-59 340 RSSQSVLYS 344 WASTRES 387 QQYYSTPRT SSNRNYLAheavy U1-61.1 257 GVSISSGGY 258 YIYYSGSTYY 316 DSESEYSSSS YWS NPSLKSNYGMDV light U1-61.1 same as U1-61.1 heavy U1-61 257 GVSISSGGY 258YIYYSGSTYY 316 DSESEYSSSS YWS NPSLKS NYGMDV light U1-61 341 RASQTISSYL359 AASSLQG 377 QQSYSNPLT N  heavy U1-62 247 GYSFTSYWI 273 IIYPGDSDTR317 QMAGNYYY G YSPSFQG GMDV light U1-62 342 RASQSVISIY 347 GASSRAT 388QQYGSSPCS LA 

1-50. (canceled)
 51. A method of treating a cancer associated with HER-3in a subject, comprising administering to the subject a first agent anda second agent, wherein said first agent is an isolated binding proteinwhich binds to HER-3, comprising: (a) a heavy chain amino acid sequencethat comprises a CDRH1 having the sequence of SEQ ID NO:236, a CDRH2having the sequence of SEQ ID NO:258, and a CDRH3 having the sequence ofSEQ ID NO:283; and a light chain amino acid sequence that comprises aCDRL1 having the sequence of SEQ ID NO:320, a CDRL2 having the sequenceof SEQ ID NO:343, and a CDRL3 having the sequence of SEQ ID NO:360; (b)a heavy chain amino acid sequence that comprises a CDRH1 having thesequence of SEQ ID NO:236, a CDRH2 having the sequence of SEQ ID NO:258,and a CDRH3 having the sequence of SEQ ID NO:285; and a light chainamino acid sequence that comprises a CDRL1 having the sequence of SEQ IDNo:320, a CDRL2 having the sequence of SEQ ID NO:343, and a CDRL3 havingthe sequence of SEQ ID NO:360; (c) a heavy chain amino acid sequencethat comprises a CDRH1 having the sequence of SEQ ID NO:251, a CDRH2having the sequence of SEQ ID NO:278, and a CDRH3 having the sequence ofSEQ ID NO:309; and a light chain amino acid sequence that comprises aCDRL1 having the sequence of SEQ ID NO:334, a CDRL2 having the sequenceof SEQ ID NO:356, and a CDRL3 having the sequence of SEQ ID NO:381; aheavy chain amino acid sequence that comprises a CDRH1 having thesequence of SEQ ID NO:252, a CDRH2 having the sequence of SEQ ID NO:280,and a CDRH3 having the sequence of SEQ ID NO:313; and a light chainamino acid sequence that comprises a CDRL1 having the sequence of SEQ IDNO:337, a CDRL2 having the sequence of SEQ ID NO:351, and a CDRL3 havingthe sequence of SEQ ID NO:385; or (d) a heavy chain amino acid sequencethat comprises a CDRH1 having the sequence of SEQ ID NO:256, a CDRH2having the sequence of SEQ ID NO:282, and a CDRH3 having the sequence ofSEQ ID NO:315; and a light chain amino acid sequence that comprises aCDRL1 having the sequence of SEQ ID NO:340, a CDRL2 having the sequenceof SEQ ID NO:344, and a CDRL3 having the sequence of SEQ ID NO:387; andsaid second agent is cetuximab.
 52. A method of treating a cancerassociated with HER-3 in a subject, comprising administering to thesubject a first agent and a second agent, wherein said first agent is anisolated binding protein which binds to HER-3, comprising a heavy chainamino acid sequence selected from the group consisting of SEQ ID NOs:42,54, 70, 92, and 96, and a light chain, and said second agent iscetuximab.
 53. A method of treating a cancer associated with HER-3 in asubject, comprising administering to the subject a first agent and asecond agent, wherein said first agent is an isolated binding proteinwhich binds to HER-3, comprising a light chain amino acid sequenceselected from the group consisting of SEQ ID NOs:44, 56, 72, 94, and 98,and a heavy chain, and said second agent is cetuximab.
 54. The method ofclaim 51, wherein said first agent is an antigen-binding protein thatbinds to HER-3, and comprises the heavy chain amino acid sequence of SEQID NO:42 and the light chain amino acid sequence of SEQ ID NO:44. 55.The method of claim 51, wherein said first agent is an antigen-bindingprotein that binds to HER-3, and comprises the heavy chain amino acidsequence of SEQ ID NO:54 and the light chain amino acid sequence of SEQID NO:
 56. 56. The method of claim 51, wherein said first agent is anantigen-binding protein that binds to HER-3, and comprises the heavychain amino acid sequence of SEQ ID NO:70 and the light chain amino acidsequence of SEQ ID NO:72.
 57. The method of claim 51, wherein said firstagent is an antigen-binding protein that binds to HER-3, and comprisesthe heavy chain amino acid sequence of SEQ ID NO:92 and the light chainamino acid sequence of SEQ ID NO:94.
 58. The method of claim 51, whereinsaid first agent is an antigen-binding protein that binds to HER-3, andcomprises the heavy chain amino acid sequence of SEQ ID NO:96 and thelight chain amino acid sequence of SEQ ID NO:98.
 59. The method of claim51, wherein said antigen-binding protein is directed against theextracellular domain of HER-3.
 60. The method of claim 51, whereinbinding of said antigen-binding protein to HER-3 reduces HER-3-mediatedsignal transduction.
 61. The method of claim 51, wherein binding of saidantigen-binding protein to HER-3 reduces HER-3 phosphorylation.
 62. Themethod of any one of claim 51, wherein binding of said antigen-bindingprotein to HER-3 reduces cell proliferation.
 63. The method of any oneof claim 51, wherein binding of said antigen-binding protein to HER-3reduces cell migration.
 64. The method of any one of claim 51, whereinbinding of said antigen-binding protein to HER-3 increases thedownregulation of HER-3.
 65. The method of any one of claim 51, whereinsaid antigen-binding protein that binds to HER-3 is an antibody.
 66. Themethod of claim 65, wherein said antibody is a monoclonal antibody, apolyclonal antibody, a recombinant antibody, a human antibody, achimeric antibody, a multispecific antibody, or an antibody fragmentthereof.
 67. The method of claim 66, wherein said antibody fragment is aFab fragment, a Fab′ fragment, a F(ab′)2 fragment, a Fv fragment, adiabody, or a single chain antibody molecule.
 68. The method of claim66, wherein said antibody is of the IgG1-, IgG2-, IgG3- or IgG4-type.69. The method of claim 51, wherein said first agent is anantigen-binding protein that binds to HER-3, and wherein saidantigen-binding protein is coupled to an effector group.
 70. The methodof claim 69, wherein said effector group is a radioisotope orradionuclide, a toxin, or a therapeutic or chemotherapeutic group. 71.The method of claim 70, wherein said therapeutic or chemotherapeuticgroup is selected from the group consisting of calicheamicin,auristatin-PE, geldanamycin, maytansine and derivatives thereof.
 72. Amethod of treating a cancer associated with HER-3 in a subject,comprising administering to the subject a first agent and a secondagent, wherein said first agent is an antigen-binding protein that bindsto HER-3 and comprises the heavy chain amino acid sequence of SEQ IDNO:70 and the light chain amino acid sequence of SEQ ID NO:72, andwherein said second agent is cetuximab.
 73. The method of claim 51,optionally comprising administering a further therapeutic agent and/orradiation therapy.
 74. The method of claim 73, wherein the furthertherapeutic agent is an anti-neoplastic agent.
 75. The method of claim74, wherein the anti-neoplastic agent is an anti-tumor antibody or achemotherapeutic agent.
 76. The method of claim 75, wherein thechemotherapeutic agent is selected from the group consisting ofcapecitabine, anthracycline, doxorubicin, cyclophosphamide, paclitaxel,docetaxel, cisplatin, gemcitabine, and carboplatin.
 77. The method ofclaim 51, wherein said first agent and said second agent areadministered by intravenous, subcutaneous, intramuscular or oraladministration.
 78. The method of claim 51, wherein said cancer isselected from the group consisting of breast cancer, ovarian cancer,prostate cancer, colon cancer, renal cancer, lung cancer, pancreaticcancer, epidermoid carcinoma, fibrosarcoma, melanoma, nasopharyngealcarcinoma, and squamous cell carcinoma.
 79. The method of claim 78,comprising administering said first agent at a dose of about 1 to about20 mg/kg body weight, at least once every 6 weeks.
 80. The method ofclaim 78, comprising administering said second agent at a dose of about1 to about 20 mg/kg body weight, at least once every 6 weeks.
 81. Themethod of claim 78, further comprising, prior to the administering,using a method that comprises analysis of a predictive marker to selecta subject having a disease associated with HER-3.
 82. The method ofclaim 78, further comprising, after the administering, monitoring thetherapeutic outcome.